Exercise induced eosinophil responses: normal cell counts with a marked decrease in responsivenessTo the Editor,Type II inflammation is characterized by elevated blood eosinophils which makes these cells an important diagnostic and treatment target in, for instance, severe asthma. Therefore, blood eosinophil numbers are a main inclusion criterion for many clinical studies that investigated the treatment of eosinophilic asthma with anti-IL5(Rα). However, there is no consensus on cut-off values for blood eosinophils during inclusion, as evidenced by a high variability between studies, ranging from 150 to 400 cells/µL. Moreover, the range of blood eosinophils in a healthy population, without confounding factors for increased blood eosinophils, is 30 – 330 cells/µL in males and 30 – 310 cells/µL in females. This implies that the cut-off values used for clinical studies greatly overlap with blood eosinophil counts that are found in the healthy population. This inherently poses a problem as eosinophil blood counts seem to be inadequate to use for diagnosing eosinophilic disease.This overlap in eosinophil counts between patients and the healthy population limits the application of eosinophil numbers for discriminating between health and several inflammatory diseases. A more promising approach in diagnosing eosinophilic disease is to combine eosinophil numbers with their activation status. Unfortunately, there is surprisingly little evidence that blood eosinophil counts correlate with their activation status and/or responsiveness in vivo in disease. This lack of correlation can be caused by ex vivo activation and/or their homing to the lung leaving behind non-activated cells in the blood.To circumvent ex vivo activation, we analyzed blood eosinophils activation status directly after venipuncture with a fast, automated, point-of-care, mobile flow cytometer (AQUIOS CL, Beckman Coulter). As exercise can be used as a model to modulate eosinophil numbers in a healthy setting, we studied whether eosinophil blood counts correlate with their activation status and their responsiveness to formyl peptides in a cohort of long-distance runners participating in a mass-participation trail run (22, 29 or 43 km). The study was approved by medical research ethics committee Oost-Nederland (NL79864.091.22). After written informed was obtained, venous blood samples were collected from 35 athletes before, directly after and 24 hours after exercise. The eosinophil activation status was assessed by combining automated flow cytometry with a 5-dimensional algorithm-based gating.An acute leukocytosis with eosinopenia was present directly post-exercise, which is in agreement with previous research. These numbers normalized 24 hours after exercise (figure 1A, 1C ). Compared to before exercise, eosinophils showed a more activated phenotype (increased CD11b and decreased CD62L) directly after exercise which also normalized within 24 hours. In marked contrast to acute inflammation, such as caused by SARS-CoV2 infection, this eosinopenia directly after exercise did not lead to refractoriness to fNLF-stimulation. However, after the normalization of eosinophil counts 24 hours after exercise, the eosinophils were refractory for activation by fNLF (figure 1B, 1C ). This clearly showed a complete dissociation between blood eosinophil numbers and their relative activation status.Our results illustrate that the eosinophil blood compartment is not adequately characterized by solely counting cell numbers (‘quantity’) as normalized numbers do not necessarily reflect normalization of their activation status (‘quality’). This finding is not limited to measuring the state of type II immunity in eosinophilic disease, but probably also applies to other infectious/inflammatory conditions and to non-pathological settings. Our data call for a re-evaluation of using blood eosinophil counts as an adequate representation of the eosinophil compartment’s state. Until recently, determining the activation status of the eosinophil compartment was complicated by ex vivo artifacts already starting at the moment of venipuncture. Now with the availability of fast, automated, point-of-care flow cytometry, it is feasible to measure both the quantity and quality of eosinophils in a wide scope of health and disease settings.
Background Drug hypersensitivity reactions (DHRs) to platinum-based drugs are heterogenous and restrict their access, and drug desensitization (DD) has provided a ground-breaking procedure for their re-introduction, although the response is heterogeneous. We aimed to identify the phenotypes, endotypes and biomarkers of reactions to carboplatin and oxaliplatin and their response to DD. Methods Seventy-nine patients presenting with DHRs to oxaliplatin (N=46), and carboplatin (N=33) were evaluated at the Allergy Departments of two tertiary care hospitals in Spain. Patient symptoms, skin testing, biomarkers, and outcomes of 267 DDs were retrospectively analyzed. Results Oxaliplatin-reactive patients presented with type I (74%), cytokine release reaction (CRR) (11%), and mixed (Mx) (15%) phenotypes. In contrast, carboplatin reactive patients presented with predominantly type I (85%) and Mx (15%) but no CRRs. Out of 267 DDs, breakthrough reactions (BTRs) to oxaliplatin occurred twice as frequently as carboplatin (32% versus 15%; p<0.05). Phenotype switching from type I to another phenotype was observed in 46% of oxaliplatin DDs compared to 21% of carboplatin DDs. Tryptase was elevated in type I and Mx reactions, and IL-6 in CRR and Mx, indicating different mechanisms and endotypes. Conclusion Carboplatin and oxaliplatin induced three different types of reactions with defined phenotypes and endotypes amendable to DD. Although most of the initial reactions for both were type I, oxaliplatin presented with unique CRR reactions. During DD, carboplatin reactive patients presented mostly type I BTR, while oxaliplatin reactive patients frequently switched from type I to CRR, providing a critical difference and the need for personalized DD protocols.
Sputum cytokines associated with raised FeNO after anti-IL5 biologic therapy in severe asthma.To the Editor,Biomarkers such as circulating absolute eosinophil count, % of eosinophils in sputum, and fraction of exhaled nitric oxide (FeNO) are predictors of response to anti-inflammatory therapy for asthma. Failure to normalize FeNO with high doses of corticosteroids are likely to be related to cytokines and chemokines such as IL-5, IL-4, IL-13, eotaxin and TARC derived from eosinophils and other Th2 cells, and alarmins such as TSLP and IL-33 from sources such as the airway epithelium(1). All Anti-IL5 biologics suppress eosinophils in sputum. However benralizumab (anti-IL5RMab) has greater effect in the severe prednisone-dependent patients than mepolizumab/reslizumab (anti-IL5 neutralizing Mab)(2,3). While raised eosinophil count is a predictor of clinical response to anti-IL5 biologics, raised FeNO is often not(4). However, they reduce FeNO to variable levels(4,5) suggesting that FeNO is partly regulated by cytokines derived from eosinophils in the airway(1). The cytokines in sputum associated with raised FeNO in prednisone-dependent severe asthmatics treated with effective anti-eosinophil drugs are not known.In this retrospective observational study, we measured cytokines in sputum using an automated ELISA reader (EllaTM, Protein Simple, R&D Systems, BioTechne, Minneapolis) at baseline and after 4 months of treatment with either benralizumab or mepolizumab/reslizumab and compared the levels of cytokines in those whose FeNO remained high after treatment. Raised FeNO was defined by FeNO >40ppb and an increase of at least 16ppb from pre-treatment value. The study was approved by Hamilton Integrated Research Ethics Board (#11227, 5037), and all patients gave written informed consent. The cytokines assayed were IL-5, IL-4 and IL-13 (Th2 inflammation) and IL-1β, IL-6, IL-10, IL-12p70, IL-15 IL-17A, IL-18, IL-33, IFNγ and TNFα (Th1/Th17 inflammation, Table e3). Details of baseline demographics, methods and statistics are shown in the online supplement.Paired measurements were made in 30 patients who received benralizumab, and 10 each who received mepolizumab/reslizumab. Overall, as previously reported(5), FeNO levels were not significantly reduced by anti-IL5 treatment (median FeNO pre-treatment 29 [5-156] vs FeNO post treatment 37 [6-280]; p=0.25; Figure e1). This change in FeNO did not correlate with a reduction of sputum eosinophils (r=-0.24; p=0.16).Among 15 patients, FeNO remained raised after treatment (Table 1). On average, IL-4 and IL-13 were the only cytokines significantly higher in the sputum of these patients compared to those in whom the FeNO values normalized (Figure 1, Figure e2). Within this group, there were patients with raised IL-4 (31%) and IL-13 (15%) and those with normal IL-4/IL-13. A small proportion of those with normal IL4/13 had raised levels of IL-18 or IL-1β (20%). Residual eosinophilic airway inflammation was significantly more present in patients with raised FeNO (30.8% vs 8.1%; P=0.04; Table e2). Patients with raised FeNO remained to have poor asthma control with an ACQ>1.5, however this did not significantly differ from those with a normalized FeNO (ACQ 1.7±0.9 vs 1.4±1.1; P=0.36; Table 1).This study, despite its limitation of retrospective design and small numbers, provide novel information on the cytokine profile in the airways of severe prednisone-dependent eosinophilic asthma patients whose FeNO remain high after anti-IL5 treatment. This is a common clinically encountered situation. Our observations suggest that IL-4/IL-13 are the cytokines most associated with this phenomenon. This may be due to the airway eosinophilia being uncontrolled or due to a non-eosinophilic source of these cytokines. However, there could be non-IL-4/IL-13 related increase in FeNO that may be due to inflammasome activation and through non-Th2 cytokine pathways that may raise the possibility of airway infections or autoimmune activation(6). This has important clinical implication. These patients may not show adequate response to switching to anti-IL4R Mab if their asthma remains uncontrolled. This needs to be evaluated prospectively.References:Couillard S, Shrimanker R, Chaudhuri R, et al. Fractional Exhaled Nitric Oxide Nonsuppression Identifies Corticosteroid-Resistant Type 2 Signaling in Severe Asthma. Am J Respir Crit Care Med 2021; 204: 731-734.Mukherjee M, Forero DF, Tran S, Boulay ME, et al. Suboptimal treatment response to anti-IL-5 monoclonal antibodies in severe eosinophilic asthmatics with airway autoimmune phenomena. Eur Respir J 2020 Oct 8;56(4):2000117. doi: 10.1183/13993003.00117-2020.Mukherjee M, Bhalla A, Venegas-Garrido C, et al. Benralizumab attenuates blood and airway eosinophilia in severe asthmatics with inadequate response to anti-IL-5 neutralizing antibodies [abstract]. Eur Respir J 2022; 60 (suppl 66): 3994; DOI: 10.1183/13993003.Hearn AP, Kavanagh J, d’Ancona G, et al. The relationship between Feno and effectiveness of mepolizumab and benralizumab in severe eosinophilic asthma. J Allergy Clin Immunol Pract 2021; 9: 2093-2096.e1.Nair P, Kjarsgaard M, Armstrong S, Efthimiadis A, O’Byrne PM, Hargreave FE. Nitric oxide in exhaled breath is poorly correlated to sputum eosinophils in patients with prednisone-dependent asthma. J Allergy Clin Immunol 2010; 126: 404-6.Donnelly LE, Barnes PJ. Expression and regulation of inducible nitric oxide synthase from human primary airway epithelial cells. Am J Respir Cell Mol Biol 2002; 26: 144-51.AuthorsPieter-Paul Hekking 1,2, Kayla Zhang1, Carmen Paz Venegas Garrido 1, Raquel Lopez-Rodriguez 1,3, Melanie Kjarsgaard1, Manali Mukherjee 1, Parameswaran Nair 11. Division of Respirology, Department of Medicine, St. Joseph’s Healthcare Hamilton, McMaster University, Hamilton, Ontario, Canada.2. Department of Respiratory Diseases, STZ Centre of Excellence for Asthma & COPD, Franciscus Gasthuis & Vlietland Hospital, Rotterdam, the Netherlands.3. Department of Allergy, Lucus Augusti Hospital, Lugo, SpainCorrespondenceDr Parameswaran NairFirestone Institute for Respiratory HealthSt Joseph’s Healthcare Hamilton50 Charlton Avenue EastHamilton, Ontario, L8N4A6, CanadaTel: 905-522-1155 x 35044Fax: 905-521-6183E-mail: email@example.com
Background: Increasing evidence are available about the presence of increased serum concentration of Immunoglobulin (Ig) Free Light Chains (FLCs) in both atopic and non-atopic inflammatory diseases, including severe asthma, providing a possible new biomarker of disease, disease severity and also an alternative approach to the treatment. Methods: We analyzed clinical and laboratory data, including FLCs, obtained from a cohort of 79 asthmatic subjects, clinically classified into different GINA steps. A control group of 40 age-matched healthy donors (HD) was considered. Particularly, HD have been selected according to the absence of monoclonal components (in order to exclude paraproteinemias), were tested for total IgE (that were in the normal ranges) and were negative for aeroallergens specific IgE. Moreover, no abnormality of common inflammatory markers (i.e. erythrocyte sedimentation rate, C-reactive protein) was detectable. Results: FLC-k levels were significantly increased in the asthmatic population, compared to the control group. Despite the absence of statistically significant differences in FLC-λ levels, the FLC-k/FLC-λ ratio displayed remarkable differences between the two groups. A positive correlation between FLC-κ and FLC-λ levels was found. FLC- λ level displayed a significant negative correlation with the FEV1 value. Moreover, the FLC-κ /FLC- λ ratio was negatively correlated with the SNOT-22 score and a positive correlation was observed between FLCs and Staphylococcus Aureus IgE enterotoxins sensitization. Conclusions: Our findings confirmed the role of FLCs in asthma as a potential biomarker in an inflammatory disease characterized by different endotypes and phenotypes. In particular, FLC-κ and FLC-k/FLC-λ ratio could be a qualitative indicator for asthma, while FLC-λ levels could be a quantitative indicator for disease severity.
Abstract: Background: The European Academy of Allergy and Clinical Immunology’s (EAACI) is updating the Guidelines on Food Allergy Diagnosis. We aimed to undertake a systematic review of the literature with meta-analyses to assess the accuracy of diagnostic tests for IgE-mediated food allergy. Methods: We searched three databases (Cochrane CENTRAL (Trials), MEDLINE (OVID) and Embase (OVID)) for diagnostic test accuracy studies published between 1 st October 2012 and 30 th June 2021 according to a previously published protocol (CRD42021259186). We independently screened abstracts, extracted data from full-texts, and assessed risk of bias with QUADRAS 2 tool in duplicate. Meta analyses were undertaken for food-test combination where 3 or more studies were available. Results: 149 studies comprising 24,489 patients met the inclusion criteria and were generally heterogeneous. 60.4% of studies were in children ≤12 years of age, 54.3% undertaken in Europe, ≥95% conducted in a specialized pediatric or allergy clinical setting and all included oral food challenge in at least a percentage of enrolled patients, in 21.5% DBPCFC. Skin prick test (SPT) with fresh cow’s milk and raw egg had high sensitivity (90% and 94%) for milk and cooked egg allergies. Specific IgE to individual components had high specificity: Ara h 2 had 92%, Cor a 14 95%, Ana o 3 94%, casein 93%, ovomucoid 92/91% for the diagnosis of peanut, hazelnut, cashew, cow’s milk and raw/cooked egg allergies, respectively. BAT was highly specific for the diagnosis of peanut (90%) and sesame (93%) allergies. Conclusions: SPT and specific IgE to extracts had high sensitivity whereas specific IgE to components and BAT had high specificity to support the diagnosis of individual food allergies. PROSPERO registration: CRD42021259186 Funding: European Academy of Allergy (EAACI).
Background: IgE-mediated cow’s milk allergy (IgE-CMA) is one of the first allergies to arise in early childhood and may result from exposure to various milk allergens, of which β-lactoglobulin (BLG) and casein are the most important. Understanding the underlying mechanisms behind IgE-CMA is imperative for the discovery of novel biomarkers and the design of innovative treatment and prevention strategies. Methods: We report a longitudinal in vivo murine model, in which 2 mice strains (BALB/c and C57Bl/6) were sensitized to BLG using either cholera toxin or an oil emulsion (n=6 per group). After sensitization, mice were challenged orally, their clinical signs monitored, antibody (IgE and IgG1) and cytokine levels (IL-4 and IFN-γ) measured, and fecal samples subjected to metabolomics. The results of the murine models were further supported by fecal microbiome-metabolome data from our population of IgE-CMA (n=24) and healthy (n=23) children (Trial: NCT04249973), on which polar metabolomics, lipidomics and 16S rRNA metasequencing were performed. In vitro gastrointestinal digestions and multi-omics corroborated the microbial origin of proposed metabolic changes. Results: During sensitization, we observed multiple microbially derived metabolic alterations, most importantly bile acid, energy and tryptophan metabolites, that preceded allergic inflammation. The latter was reflected in a disturbed sphingolipid metabolism. We confirmed microbial dysbiosis, and its causal effect on metabolic alterations in our patient cohort, which was accompanied by metabolic signatures of low-grade inflammation. Conclusion: Our results indicate that gut dysbiosis precedes allergic inflammation and nurtures a chronic low-grade inflammation in children on elimination diets, opening important new opportunities for future prevention and treatment strategies.
Background Allergic rhinitis (AR) is one of the most common chronic diseases worldwide. There are limited prospective long-term data regarding persistency and remission of AR. The objective of this study was to investigate the natural course of pollen-induced AR (pollen-AR) over 20 years, from childhood into early adulthood. Methods Data from 1137 subjects in the Barn/Children Allergi/Allergy Milieu Stockholm Epidemiologic birth cohort (BAMSE) with a completed questionnaire regarding symptoms, asthma, treatment with allergen immunotherapy (AIT) and results of allergen-specific IgE for inhalant allergens at 4, 8, 16 and 24 years were analysed. Pollen-AR was defined as sneezing, runny, itchy, or blocked nose; and itchy or watery eyes when exposed to birch and/or grass pollen in combination with allergen-specific IgE ≥0.35kU A/l to birch and/or grass. Results Approximately 75% of children with pollen-AR at 4 or 8 years had persistent disease up to 24 years, and 30% developed asthma. The probability of persistency was high already at low levels of pollen-specific IgE. The highest rate of remission from pollen-AR was seen between 16 and 24 years (21.5%), however the majority remained sensitized. This period was also when pollen-specific IgE-levels stopped increasing and the average estimated annual incidence of pollen-AR decreased from 1.5% to 0.8% per year. Conclusion Children with pollen-AR are at high risk of persistent disease for at least 20 years. Childhood up to adolescence seems to be the most dynamic period of AR progression. Our findings underline the close cross-sectional and longitudinal relationship between sensitization, AR, and asthma.
Background Thymic stromal lymphopoietin (TSLP), a pleiotropic cytokine mainly expressed by epithelial cells, plays a key role in asthma pathobiology. In humans, TSLP exists in two variants: the long form TSLP (lfTSLP) and a shorter TSLP isoform (sfTSLP), overlapping the lfTSLP C-terminus. Macrophages (HLMs) and mast cells (HLMCs) are in close proximity in the human lung and play central roles in different asthma phenotypes. Methods Immunofluorescence and Western blot were employed to localize intracellular TSLP. Limited proteolysis and mass spectrometry allowed the identification of cleavage sites of TSLP caused by tryptase and chymase. ELISA assays were employed to measure TSLP and VEGF-A. Results TSLP was detected in highly purified (≥ 99%) macrophages isolated from human lung and subcellularly localized in the cytoplasm by confocal microscopy and Western blot. IL-4 and lipopolysaccharide induced the release of TSLP from HLMs. HLMCs contain and release tryptase and chymase that specifically cleaved TSLP. Mass spectrometric analyses of TSLP treated with tryptase showed the production of 1-97 and 98-132 fragments. Chymase treatment of TSLP generated two peptides 1-36 and 37-132. HLM activation by lfTSLP induced VEGF-A, the most potent angiogenic factor, release. The four TSLP fragments generated by tryptase and chymase failed to activate HLMs. sfTSLP neither activated HLMs nor interfered with activating property of lfTSLP on HLMs. Conclusions Given the close proximity between mast cells and macrophages in the human lung, our results illuminate a new circuit between HLMs and mast cells. These findings have potential relevance in understanding novel aspects of asthma pathobiology.
Background Activation of mast cells through IgE results in secretion and shedding of mast cell proteins and in vivo models suggest that these processes are governed by IgE antibody affinity. Methods We passively sensitized cultured primary human mast cells with recombinant human IgE clones with either high or low affinity for Der p 2, with a 200-fold affinity difference, and activated them with recombinant allergen. Activation was assessed by CD63 upregulation and PGD 2 secretion. Supernatants collected from mast cells activated for 0, 3, 6 and 24 hours were assessed for PGD 2 and inflammatory mediators on the OLINK platform at repeated time points. Results CD63 upregulation and PGD 2 synthesis scaled with affinity, as did secretion of cytokines like IL-8 and IL-13. Secretion of chemokines like CCL3 and CCL4 appeared to depend less on affinity, whereas shedding of surface markers CD40, SLAMF4 and CD5, and secretion of intracellular markers SIRT2 and CASP-8, were elevated by stimulation through low affinity IgE compared with high affinity IgE, illustrating differential responses dependent on the affinity of IgE. Conclusion Cytokine secretion and shedding of surface receptors of sensitized, cultured primary human mast cells is differentially regulated depending on the affinity of IgE for the Der p 2 allergen and may shape the chronic response to repeated allergic activation.
Background: Modelling atopic dermatitis (AD) in vitro is paramount to understand the disease pathophysiology and identify novel treatments. Previous studies have shown that the Th2 cytokines IL-4 and IL-13 induce AD-like features in keratinocytes in vitro. However, it has not been systematically researched whether the addition of Th2 cells, their supernatants or a 3D structure are superior to model AD compared to simple 2D cell culture with cytokines. Methods: For the first time, we investigated what in vitro option most closely resembles the disease in vivo based on single-cell RNA sequencing data (scRNA-seq) obtained from skin biopsies in a clinical study and published datasets of healthy and AD donors. In vitro models were generated with primary fibroblasts and keratinocytes, subjected to cytokine treatment or Th2 cell cocultures in 2D/3D. Gene expression changes were assessed using qPCR and Multiplex Immunoassays. Results: Of all cytokines tested, incubation of keratinocytes and fibroblasts with IL-4 and IL-13 induced the closest in vivo-like AD phenotype which was observed in the scRNA-seq data. Addition of Th2 cells to fibroblasts failed to model AD due to the downregulation of ECM-associated genes such as POSTN. While keratinocytes cultured in 3D showed better stratification than in 2D, changes induced with AD triggers did not better resemble AD keratinocyte subtypes observed in vivo. Conclusions: Taken together, our comprehensive study shows that the simple model using IL-4 or IL-13 in 2D most accurately models AD in fibroblasts and keratinocytes in vitro, which may aid the discovery of novel treatment options.
Transcription therapy is an emerging approach that centers on identifying the factors associated with the malfunctioning gene transcription machinery that causes diseases and controlling them with designer agents. Until now, small molecule drugs targeting the epigenetic enzymes and critical signaling pathways have been the primary research focus in therapeutic gene modulation. However, nucleic acid-based small molecules have gained popularity in recent years as they could be pre-designed on demand to achieve operative control over the dynamic transcription machinery that governs how the immune system responds to diseases. Pyrrole-imidazole polyamides (PIPs) are well-established DNA-based small molecule gene regulators that overcome the limitations of their conventional counterparts owing to their sequence-targeted specificity, versatile regulatory efficiency and biocompatibility. Here, we emphasize the rational design of PIPs, their functional mechanism and their potential as targeted transcription therapeutics for diseases by regulating the immune response. Furthermore, we also discuss the challenges and foresight of this approach in personalized immunotherapy in precision medicine.
Background Respiratory syncytial virus (RSV) infection in infants is a major cause of viral bronchiolitis and hospitalisation. We have previously shown in a murine model that ongoing infection with the gut helminth Heligmosomoides polygyrus ( H. polygyrus) protects against RSV infection through type I interferon (IFN-I) dependent reduction of viral load. Yet, the cellular basis for this protection has remained elusive. Given that recruitment of mononuclear phagocytes to the lung is critical for early RSV infection control, we assessed their role in this coinfection model. Methods Mice were infected by oral gavage with H. polygyrus. Myeloid immune cell populations were assessed by flow cytometry in lung, blood and bone marrow throughout infection and after secondary infection with RSV. Monocyte numbers were depleted by anti-CCR2 antibody or increased by intravenous transfer of enriched monocytes. Results H. polygyrus infection induces bone marrow monopoiesis, increasing circulatory monocytes and lung mononuclear phagocytes in a IFN-I signalling dependent manner. This expansion causes enhanced lung mononuclear phagocyte counts early in RSV infection that may contribute to the reduction of RSV load. Depletion or supplementation of circulatory monocytes prior to RSV infection confirms that these are both necessary and sufficient for helminth induced antiviral protection. Conclusions H. polygyrus infection induces systemic monocytosis contributing to elevated mononuclear phagocyte numbers in the lung. These cells are central to an anti-viral effect that reduces the peak viral load in RSV infection. Treatments to promote or modulate these cells may provide novel paths to control RSV infection in high risk individuals.
Early expansion of allergen-responsive LAP+ B regulatory cells in allergic rhinitis but not in allergic asthma subjects during allergen immunotherapyAstrid L. Voskamp1, Nicolette W. de Jong2, Simon P. Jochems1, Arifa Ozir-Fazalalikhan1, Luciën E.P.M. van der Vlugt1, Koen A. Stam1, Gert-Jan Braunstahl3,4, Gertrude M. Möller5, Roy Gerth van Wijk2, Hermelijn H. Smits11Department of Parasitology, Leiden University Center for Infectious Diseases (LU-CID), Leiden University Medical Center; Leiden, The Netherlands.2Dept of Internal Medicine, Section Allergology and Clinical Immunology, Erasmus University Medical Center; Rotterdam, The Netherlands.3Department of Pulmonology, Franciscus Gasthuis and Vlietland, Rotterdam, 3045 PM, the Netherlands.4Department of Pulmonology, Erasmus Medical Center, Rotterdam, 3015 GD, the Netherlands.5Netherlands Center of Clinical Occupational Medicine, The Netherlands.
Background: Respiratory birch pollen allergy and associated oral allergy syndrome affect more than 150 million people. IgE cross-sensitization to major birch pollen allergen Bet v 1 and pathogenesis-related (PR10) plant food allergens is responsible for the pollen-food allergy syndrome. Methods: We designed a recombinant protein, AB-PreS, consisting of non-allergenic peptides derived from the IgE binding sites of Bet v 1 and the cross-reactive apple allergen, Mal d 1, fused to the PreS domain of HBV surface protein as immunological carrier. AB-PreS was expressed in E. coli and purified by chromatography. The allergenic activity of AB-PreS was tested using sera and basophils from birch pollen patients allergic. The protective effect of AB-PreS was assessed by inhibition ELISA test using sera allergic patients and from immunized rabbits. Results: IgE-binding experiments and basophil activation test revealed the hypoallergenic nature of AB-PreS. IgG antibodies induced by 5 injections with AB-PreS inhibited allergic patients’ IgE binding to Bet v 1 and Mal d 1 better than did IgG induced by up to 30 injections of six licensed birch pollen allergen extract-based vaccines. Additionally, immunization with AB-PreS induced HBV-specific antibodies potentially protecting the infection. Conclusion: The recombinant AB-PreS-based vaccine is hypoallergenic, safe and superior to currently registered allergen extract-based vaccines for the treatment of the birch pollen food allergy syndrome.
A consensus protocol for the Basophil Activation Test for multicenter collaboration and External Quality AssuranceAuthors: Pascal, M# 1, Edelman SM#2, Nopp, A#3, Möbs, C4, Geilenkeuser, WJ5, Knol, EF6, Ebo, DG7, Mertens C7, Shamji, MH8, Santos, AF9,10, Patil, S11, Eberlein, B*12, Mayorga, C*13, Hoffmann HJ14*Affiliations1 Immunology Department, Centre de Diagnòstic Biomèdic, Hospital Clínic de Barcelona, Barcelona, Spain; Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Spain.2 Skin and Allergy Hospital, Helsinki University Central Hospital, Helsinki, Finland, present address Aimmune Therapeutics, Finland3 Department of Clinical Science and Education, Karolinska Institutet, Södersjukhuset, and Sachs´ Children and Youth Hospital, Södersjukhuset, Stockholm, Sweden4 Department of Dermatology and Allergology, Philipps-Universität Marburg, Marburg, Germany5 Reference Institute for Bioanalytics, Bonn, Germany6 Center of Translational Immunology and Dermatology/Allergology, University Medical Center Utrecht, Utrecht, The Netherlands.7 Faculty of Medicine and Health Sciences, Department of Immunology-Allergology- Rheumatology, University of Antwerp, Antwerp, Belgium8 National Heart and Lung Institute, Imperial College London, UK and NIHR Imperial Biomedical Research Centre, UK9 Department of Women and Children’s Health (Pediatric Allergy) & Peter Gorer Department of Immunobiology, Faculty of Life Sciences and Medicine, King’s College London, London, United Kingdom10 Children’s Allergy Service, Evelina London Children’s Hospital, Guy’s and St Thomas’ Hospital, London, United Kingdom11 Division of Allergy and Immunology, Departments of Medicine and Pediatrics, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States12 Department of Dermatology and Allergy Biederstein, School of Medicine, Technical University Munich, Munich, Germany13 Allergy Clinical Unit, Hospital Regional Universitario de Málaga and Allergy Research Group, Instituto de Investigación Biomédica de Málaga-IBIMA-BIONAND, Malaga, Spain;14 Department of Clinical Medicine, Aarhus University, Department of Respiratory Diseases and Allergy, Aarhus University Hospital, Denmark# shared first authors, * shared senior authorsCOIM Pascal, SM Edelman, A Nopp, C Möbs, EF Knol, SU Patil and C Mayorga have no conflict of interest regarding this work. B Eberlein received methodological and technical support from the company BUEHLMANN Laboratories AG (Schönenbuch, Switzerland) outside the submitted work. Dr Hoffmann reports grant from the Innovation Fund of Denmark, outside the submitted work. Dr Shamji reports grants awarded to institution from the Immune Tolerance Network, UK Medical Research Council, Allergy Therapeuitics, LETI Laboratories, Revolo biotherapeutics and Angany Inc. He has received consulting fees from Bristol Meyers Squibb and lecture fees from Allergy Therapeutics and LETI laboratories, all outside the submitted work. Dr. Santos reports grants from Medical Research Council (MR/M008517/1; MC/PC/18052; MR/T032081/1), Food Allergy Research and Education (FARE), the NIH, Asthma UK (AUK-BC-2015-01), the Immune Tolerance Network/National Institute of Allergy and Infectious Diseases (NIAID, NIH) and the NIHR through the Biomedical Research Centre (BRC) award to Guy’s and St Thomas’ NHS Foundation Trust, during the conduct of the study; speaker or consultancy fees from Thermo Scientific, Nutricia, Infomed, Novartis, Allergy Therapeutics, IgGenix, Stallergenes, Buhlmann, as well as research support from Buhlmann and Thermo Fisher Scientific through a collaboration agreement with King’s College London, outside the submitted work. Dr Geilenkeuser is an employee of Referenizinstitut für Bioanalytik, DE that provided logistic assistance and reagent support for the study.To the editorThe basophil activation test (BAT) has significant potential as a diagnostic tool to better phenotype and manage patients with IgE-mediated allergies, so that only a small proportion of patients need to be challenged. Sample, reagent, laboratory procedure, analysis protocols, and population characteristics can influence BAT performance (1,2). Regulatory approval and clinical implementation require extensive standardization of laboratory protocols, cytometer settings, and results interpretation (3). European national authorities require External Quality Assurance (EQA) of the performance of modern diagnostic laboratories by agencies independent of test suppliers to meet ISO 15189:2012, 15189:2013 and 9001:2015.Based on an online survey among 59 responding European laboratories performing BAT in 2017 (4,5) (Online Supplement; Results of the online survey), a Task Force was launched in 2018 to create the basis for a BAT-EQA. Round Robins (RR) were organized with seven shipments of 2 donors each to 7-10 European centers with overnight courier service from Bonn, DE. To minimize variation, prior to shipment, blood basophils were activated with 1 ul FcεRI antibody/ml of blood and stabilized with 0.2 mL Transfix (Cytomark, UK) per mL of blood to stabilize activated basophils up to 48 hours for staining (6). Fresh blood was included for stimulation and staining at the participating laboratory sites.We met after the third shipment to reach consensus on a protocol for BAT (Online Supplement; Proposed SOP for in house BAT). The threshold set on an unstimulated control sample was determined empirically on an independent data set as equal or greater than 2.5% with ROC curves based on data from patients with hypersensitivity to amoxicillin and patients with peanut allergy, (Online supplement, tables S1 and S2). This proposal did not find universal consensus among the authors.Data analysis started with identification of the relevant region in a scatter plot, followed by identification of basophils with the relevant markers, for instance, using low SSC and CD193 only or CD193 and CD123. Finally, the threshold was set at 2.5% of CD63 expression on resting basophils (Figure 1A). >5% CD63+basophils above that threshold in an activated sample was considered a positive response. This setting was used to obtain the percentage of CD63+ cells in centrally preactivated and locally activated blood samples; however, it was not adopted in all labs. Data from participating labs analyzed with their proprietary and the above standardized analysis compared well (online supplement, figure S4).The first two RR were used to establish coherence between participating laboratories. Data from RR3–RR7 were comparable. The standard deviation of activation measured at all participating centers was 16.8% in preactivated blood (Figure 1B) compared with 49.2% for samples activated and analyzed locally, illustrating the utility of using preactivated blood for EQA. Shipment to Málaga took 48h, and local activation of blood basophils was consistently suboptimal, consistent with a preliminary round robin from 2012, where the clinical outcome was robust up to 24 h. Centrally activated basophils performed as well in Málaga as in other centers.EQA for BAT is critical to facilitate routine implementation of this assay in the field of in vitro allergy diagnostics. The variability of the responses to our survey highlighted the importance and need for multicenter validation. Full validation and standardization of the BAT protocol and analysis is essential and possible for setting the grounds for controlled multicenter research studies as well as EQA. The BAT-EQA Task Force provides a standard operating protocol (Online supplement; Proposed SOP for in house BAT) and reference materials for the test to standardize and enhance the accuracy of BAT for both clinical and research collaborations and EQA.
Drug reaction with eosinophilia and systemic symptoms (DRESS), also known as Drug-induced hypersensitivity syndrome (DIHS), is a rare but severe delayed-type drug hypersensitivity reaction [(#ref-0001)1]. Its reported incidence ranges between 2 and 5 cases per million per year and the mortality between 5 and 10% [(#ref-0002)2]. DRESS is characterized by the occurrence of an extensive rash with face edema, lymphadenopathy and fever and organ damage, all of which seems to result from massive drug-directed T cell response and associated eosinophilia. DRESS is a complex condition, its clinical presentation varies depending on the cutaneous manifestation(s), affected target organ(s) and reaction severity. The diagnosis of DRESS is further challenged by the clinical overlay with autoimmune, infectious and lymphoproliferative conditions, which have to be considered in the differential diagnosis (Table 1). Eosinophilia is detected in only 80 % of DRESS patients and can be masked by e.g. the administration of systemic glucocorticoids (GCS). Furthermore, there are various differences in the DRESS diagnostic criteria (Table 1) developed by the Japanese SCAR (JSPS) [(#ref-0003)3] and RegiSCAR [(#ref-0004)4] groups, the most notable being the inclusion of herpes viremia in the criteria developed by the JSPS. All these clinical challenges underline the importance of a systematic and comprehensive approach when encountering a patient with suspected DRESS. Based on the most recent literature and our clinical expertise, we therefore suggest the medical algorithm depicted in Figure 1. DRESS should be evoked as a differential diagnosis in patients with a rash suspected to be drug-related and associated with head-and-neck edema [(#ref-0005)5]. Clinical history-taking is a critical element to consolidate or discard a drug-related etiology: most importantly, this should explore the dynamics of both possible DRESS clinical symptoms and drug exposure(s) (date of onset, way and length of administration, previous exposures / reactions). A long drug exposure prior to disease onset, i.e. 2-8 weeks, is indicative for DRESS rather than other drug hypersensitivities – but the duration may vary depending on the causative drug. A thorough clinical examination, basic laboratory work-up, electrocardiogram, and - if a rash is present - a skin biopsy should also be performed. If the clinical presentation and drug exposure history substantiate the DRESS diagnosis, additional investigations should be performed depending on the suspected target organ damage (cf. case “complementary, patient-specific work-up”). Once the diagnosis is established, a severity assessment is warranted, since DRESS can range from mild forms with very limited organ damage to fulminant ones, e.g. characterized by (multi-)organ failure. There are no consensual severity scoring. In this algorithm, we suggest the scoring system used in France (RCT DRESSCODE, https://clinicaltrial.gov NCT01987076).
Immune modulation is a key therapeutic tool for allergic diseases and asthma. It can be achieved in an antigen-specific way via allergen immunotherapy (AIT) or in endotype-driven approach using biologicals that target the major pathways of the type 2 (T2) immune response: IgE, IL-5 and IL-4/IL-13. COVID-19 vaccine provides an excellent opportunity to tackle the global pandemics and is currently being applied in an accelerated rhythm worldwide. It works as well through immune modulation. Thus, as there is an obvious interference between these treatment modalities recommendations on how they should be applied in sequence are expected. The European Academy of Allergy and Clinical Immunology (EAACI) gathered an outstanding expert panel under its Research and Outreach Committee (ROC). This expert panel was called to evaluate the evidence and formulate recommendation on the administration of COVID-19 vaccine in patients with allergic diseases and asthma receiving AIT or biologicals. The panel also formulated recommendations for COVID-19 vaccine in association with biologicals targeting the type 1 or type 3 immune response. In formulating recommendations, the panel evaluated the mechanisms of COVID-19 infection, of COVID-19 vaccine, of AIT and of biologicals and considered the data published for other anti-infectious vaccines administered concurrently with AIT or biologicals.
Needle-free Epicutaneous For t 2 DNA Vaccine is Effective for Preventing and Treating Biting Midge (Forcipomyia taiwana) allergy in a murine modelTo the Editor,Allergen-specific immunotherapy (ASIT) remains the only treatment capable of inducing immune tolerance to the corresponding allergen and potentially treating the root cause of the allergic disease.1 As the treatment course of protein-based vaccines for ASIT is time-consuming, an easily administered epicutaneous anti-allergic DNA-based vaccine is an attractive method, especially in light of the COVID-19 pandemic.2 We established a mouse model of biting midge allergy to test the concept of the epicutaneous DNA vaccine.The biting midge, Forcipomyia taiwana , is the most prevalent cause of biting insect allergy in Taiwan. It is a tiny hematophagous midge that attacks en masse. As many as 60% of exposed individuals develop allergic reactions to the bites.3 The midge is widely distributed throughout Taiwan and southern China. Among the identified allergens, For t 2 is the most predominant, with 75% of midge-allergic patients showing specific IgE to For t 2.4E.coli -expressed For t 2 recombinant protein (rFor t 2) was used as an allergen to sensitize and challenge the mice.5For t 2-encoding fragment (GenBank accession EU678971) was amplified by PCR. The PCR products were subcloned into pVAX1 (Life Technologies, Carlsbad, CA) . The experiments were designed using two approaches: therapeutic and prophylactic (Fig 1). Twenty-five μg For t 2 DNA was determined as the optimal dose after several dose-finding experiments (Fig S1, data not shown). For each treatment, the hair of the abdominal area of the mice was removed using a depilatory, tape-stripped, then patched with 25 μg For t 2 DNA vaccine for one hour and removed. A total of three treatments were given spaced one week apart (Fig 1 and Fig S2). For t 2 proteins were detected in the patched skin and the immune organ spleen at 24 hours and had significantly increased at 48 hours (Fig S3). Scratch bouts after rFor t 2 challenge were used as a clinical surrogate of itch. We measured total IgE and For t 2-specific IgG2a in the sera as well as mRNA and proteins of IL-13, interferon-gamma, IL-10, and FOXP3 in the culture supernatants of splenocytes after stimulation with various doses of rFor t 2 at 37℃ for 3-5 days by ELISA and real-time quantitative PCR. Histopathology of the challenged skins was examined.We found that after epicutaneous DNA vaccination, the allergen-induced itch of the mice significantly improved, and For t 2-specific IgG2a increased (Fig 2). Both mRNA and protein of IL-13, and eosinophils infiltration in the targeted skin, significantly decreased. Expression of FOXP3 mRNA increased (Fig S4-S6).This is the first study to demonstrate an epicutaneous anti-allergic DNA vaccine that is effective at both treating an established allergic condition and preventing the development of an allergic disease using biting midge allergy as a model. After epicutaneous DNA vaccination, in addition to the significant improvement in allergen-induced itch, the changes of biomarkers for allergic inflammation, including IgE, allergen-specific IgG2a, allergen-challenge-induced eosinophil infiltration in the skin, Th2 cytokines from the splenocytes, and regulatory T cell-related transcription factors, suggest that immune tolerance was induced after three patches of the epicutaneous DNA vaccine.Our data show that though the molecular weight of the For t 2 DNA vaccine is as high as 4000 base pairs, it is able to penetrate the dermal barrier and translates the corresponding protein in the targeted skin as well as the spleen of the vaccinated mice. It is possible that the DNA vaccine passes the epidermis via the hair follicles as the skin is tape-stripped before epicutaneous vaccination.6The mode of this anti-allergic epicutaneous DNA vaccine may have potential for use in other specific immunotherapies for other allergens.Mey Fann Lee1Chi Sheng Wu2 Shyh Jye Lin3Yi Hsing Chen2,4*1Department of Medical Research, Taichung Veterans General Hospital, Taichung, Taiwan2Division of Allergy, Immunology and Rheumatology, Taichung Veterans General Hospital, Taichung, Taiwan3School of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung, Taiwan4School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan