A document by Xiaoting Song. Click on the document to view its contents.

A document by Qiaoxing Liang. Click on the document to view its contents.

Background Essential training for emergency adrenaline auto-injector administration alone provides inadequate safeguard in school environments. Recent UK deaths have reinforced the urgency for embedding whole school (WS) allergy awareness to minimise risk. We document development of a practical, flexible WS Food Allergy Awareness Toolkit for UK secondary schools. Methods We used a multidisciplinary participatory action research methodology, involving successive modification and retesting of a pragmatic toolkit in 3 case study schools. A School Allergy Action Group drives WS risk assessment, helping schools gradually implement best practice policy in line with their particular needs. Additional schools self-piloted the resulting toolkit with only remote monitoring. School surveys, based on EAACI guidelines were developed to identify priorities and assess change. Results Effectiveness of the resulting process toolkit, now available online, was independently demonstrated via pre/post intervention questionnaires from 24/10 pupils with food allergy (FA) and 97/6 pupils without FA, respectively. Pearson correlational analysis showed strong negative relationships between Food Allergy Quality of Life Questionnaire (FAQLQ) at T0 and School Support (SS) at T0 (r=-0.8, p<0.01), and between SS and Self-Efficacy (SE) (r=0.73, p<0.05). Mean FAQLQ scores improved between T0 (3.3) and T1 (2.5). SE improved for those with FA (mean difference =1.0). In those without FA, SE (mean difference =0.9) and Attitudes and Knowledge (mean difference =0.7) also improved. Conclusions Full stakeholder involvement in toolkit development encourages usage and therefore improves WS community awareness; reduces risk of reactions; fosters a more accepting societal attitude; and empowers pupils with/without allergies to self-manage effectively.

Background: The SQ tree SLIT-tablet has recently been approved for treatment of tree pollen allergy. Health care workers should be provided with detailed safety data for clinical use. Objective: To assess the tolerability and safety of the SQ tree SLIT-tablet in adults and adolescents. Methods: Safety data were pooled from two phase-II and one phase-III double-blinded, randomized, placebo-controlled trials including adults and adolescents with allergic rhinitis and/or conjunctivitis treated before and during one pollen season once-daily with the SQ tree SLIT-tablet (12 SQ-Bet) or placebo. Results: The most frequently reported IMP-related AEs with 12 SQ-Bet were oral pruritis (39% of subjects) and throat irritation (29%). IMP-related AEs were mainly mild or moderate in severity, and the majority resolved without treatment and did not lead to treatment interruption/discontinuation. With 12 SQ-Bet, oral pruritus was more frequent among PFS subjects (45%) than in subjects without PFS (29%). A greater proportion of PFS subjects interrupted treatment (19%) than subjects without PFS (7%). The 12 SQ-Bet did not seem to induce an increased risk of asthma: 7 events were reported in 7 subjects with 12 SQ-Bet and 11 in 10 subjects with placebo. No differences were seen in the risk of moderate to severe IMP-related AEs regardless of age, PFS status and asthma medical history. Conclusions: The 12 SQ tree SLIT-tablet was well tolerated in tree pollen allergic subjects with no major safety concerns detected. This safety profile supports daily at-home sublingual administration once the first dose is tolerated when administered under medical supervision.

Article Type: News and Views: Groundbreaking Discoveries in ImmunologyTitle: Emollients for the prevention of atopic dermatitisAuthors: Akash Kothari1(https://orcid.org/0000-0003-1980-161X), Arielle Locke2,Thomas Eiwegger1,3,4(https://orcid.org/0000-0002-2914-7829)1Translational Medicine Program, Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada2Department of Medicine, National University of Ireland, Galway, Ireland3Division of Immunology and Allergy, Food Allergy and Anaphylaxis Program, The Department of Pediatrics, Hospital for Sick Children, Toronto, Ontario, Canada4Department of Immunology, Faculty of Medicine, University of Toronto, Toronto, Ontario, CanadaCorrespondence to: Thomas Eiwegger, MD, Division of Immunology and Allergy, Food Allergy and Anaphylaxis Program, The Department of Paediatrics, Hospital for Sick Children, 555 University Ave, Toronto, Canada, E-mail: [email protected], Tel.: +1 416-813-7654 ext. 1862Conflicts of Interest: AK and AL have nothing to disclose. TE reports to act as local PI for company sponsored trials by DBV and sub-investigator for Regeneron, holds grants from Innovation Fund Denmark, CIHR outside the submitted work. He is Co-Investigator or scientific lead in three investigator initiated oral immunotherapy trials supported by the Food Allergy and Anaphylaxis Program SickKids and serves as associate editor for Allergy. He/his lab received unconditional/in-kind contributions from Macro Array Diagnostics and an unrestricted grant from ALK. He holds advisory board roles for ALK.Financial support: This work was supported by The Hospital for Sick Children, The Food Allergy and Anaphylaxis Program at The Hospital for Sick Children, and The Dr Lorus J And Dr Margery J Milne Scholarship from Victoria University at the University of Toronto.Statement of Author Contribution: All authors critically reviewed the original articles (references 6 and 7) and wrote the News & Views: Groundbreaking discoveries in Immunology article. All authors contributed, revised, edited, and approved the final version of the manuscript as submitted and agreed to be accountable for all aspects of the work.Keywords: emollient, atopic eczema, infancyAbbreviations : food allergy, FA; filaggrin gene, FLG; atopic dermatitis, AD; transepidermal water loss, TEWL; Barrier Enhancement for Eczema Prevention, BEEP; Preventing Atopic Dermatitis and Allergies, PreventADALL

Mass cytometry reveals unique subsets of T cells and lymphoid cells in nasal polyps from patients with chronic rhinosinusitis (CRS)

Food allergy is an increasingly common disease worldwide, and is thought to be driven by an uncontrolled type 2 immune response. Current knowledge about the underlying mechanisms that initiate and promote an inappropriate immune response to dietary allergens is limited. Sensitization through the skin in early life is considered to be a key event. Food allergy results from a dysregulated type 2 response to food allergens, characterized by enhanced levels of IgE, IL-4, IL-5 and IL-13 with infiltration of mast cells, eosinophils and basophils during acute reactions. Recent data implies a possible role of innate lymphoid cells (ILCs) in driving food allergy. ILCs represent a group of lymphocytes that lack specific, recombined antigen receptors. They contribute to immune responses not only through the release of cytokines and other mediators, but also by responding to cytokines produced by activated cells in their local microenvironment. Due to their localization at barrier surfaces of the airways, gut and skin, ILCs form a link between the innate and adaptive immunity. This review summarizes recent evidences on how skin and gastrointestinal mucosal immune system contribute to both homeostasis and the development of food allergy, as well as the involvement of ILCs towards inflammatory processes and regulatory mechanisms.

Sustained milk consumption after 2 years post-Milk Epicutaneous therapy for Eosinophilic EsophagitisJonathan M. Spergel, MD, PhD1,2; Amanda B. Muir, MD2,3; Chris A. Liacouras, MD2,3;Deirdre Burke1, CRA; Megan O. Lewis, MSN, RN, CPNP1; Terri Brown-Whitehorn, MD1,2, Antonella Cianferoni, MD, PhD1,21Division of Allergy and Immunology, The Children’s Hospital of Philadelphia, PA, USA, 2Department of Pediatrics, The Perelman School of Medicine at University of Pennsylvania, Philadelphia, PA, USA, 3Division of Gastroenterology, Hepatology, and Nutrition, The Children’s Hospital of Philadelphia, PA, USAAuthor contribution:JMS-study design, writing manuscript, interpreting data; ABM-writing manuscript, study visits, data interpretation; CAL-writing manuscript, study design, interpreting data; DB-writing manuscript, regulatory coordinator, coordination and collection of data, study visits; MOL-writing manuscript, collection of data, study visits; TBW-writing manuscript, collection of data and study visits, AC-interpreting data, collection of data, study visits, regulatory itemsTo the Editor:Eosinophilic Esophagitis (EoE) is an allergic disease of the esophagus without any curative therapy. Typical symptoms of EoE are feeding difficulties, vomiting, abdominal pain and dysphagia and vary by age, with a diagnosis confirmed on esophageal biopsy with> 15 eosinophils/high power field (eos/hpf).1 The two treatment options for pediatric EoE2 are: 1) topical swallowed steroids, which is effective in inducing EoE remission in 50-90% of patients, depending on the dose, formulation and medication used; 2) dietary elimination of the causative allergen/s which is effective in 50-70% of patients with selective food elimination, and 95% with elemental diets3. Cow’s milk (CM) is the most common food allergen causing disease in up to 65% of patients.4 When either treatment is discontinued, inflammation and symptoms recur.3Epicutaneous immunotherapy (EPIT) is an investigational immunotherapy using low dose allergen exposure through the skin to induce desensitization. In the randomized controlled clinical trial,Study of Efficacy and Safety of Viaskin® Milk for CM-induced EoE (SMILEE Study) , 20 pediatric participants with CM-induced EoE were randomized to receive EPIT with Viaskin® Milk (n=15) or placebo (n=5)(details in appendix). After CM-induced EoE was confirmed, EPIT therapy was applied daily for 9 months during a CM-free period, followed by CM-containing diet for 2 months (Figure 1). At 11 months, subjects completed an upper endoscopy with biopsy to evaluate tissue eosinophilia as the primary endpoint. In the pre-defined per-protocol population (7 patients-Viaskin® Milk, 2 patients- placebo), Viaskin® Milk treated subjects had a lower number of eosinophils/high power field (eos/hpf) on biopsy (25.57 ± 31.19) compared to placebo (95 ± 63.64). After the blinded phase, 19 subjects were eligible to enroll in the open-label extension (additional 11 months of therapy) and had repeat endoscopy and biopsy. At the end of the open-label phase, 6/19 subjects had < 6 eos/hpf (32% response rate); 3/19 subjects had 7-14 eos/hpf for total response rate of 47%.5As part of routine clinical care, we continue to follow all 19 subjects who completed the open-label extension (currently 2 years after the end of Viaskin® Milk therapy) to understand whether CM continued in their diet without symptoms. Four of 5 subjects who had <6 eos/hpf after milk introduction were able to continue with approximately 2 servings of CM/day without any symptoms (Table 1). One of these patients had a clinically indicated endoscopy and biopsy that had 0 eosinophils. Two subjects, who had 6-14 eos/hpf during the study, continued to tolerate CM, including one subject who continued to have 6-14 eos/hpf on repeat endoscopy. In addition, 4 subjects who had significant symptoms ingesting CM and had > 15 eos/hpf during the initial SMILEE study were able to add CM back into their diet without having symptoms, as either baked CM (n=2) or regular CM with concomitant swallowed steroids therapy (n=2).The follow-up of this pilot study for the use of EPIT for milk-induced EoE suggests that the treatment effect can persist for 2 years after stopping therapy; six out of 7 patients in the responder and partial responder groups remain completely symptom-free while consuming an average of 2 servings/day of CM. In contrast to the current therapies of diet elimination or swallowed topical steroids where symptoms return when therapy is stopped, EPIT has demonstrated a persistent effectiveness. These findings align with EPIT’s proposed mechanism of action, by directly targeting and reprogramming the immune response to allergen.3 EPIT may induce true tolerance, as is observed in murine models, where Foxp3(+) CD25(+) CD4(+) T regulatory cells are induced and can transfer tolerance.6 Further longer-term studies are needed to examine this possibility and confirm these unique findings.Reference:1. Spergel JM, Dellon ES, Liacouras CA, et al. Summary of the updated international consensus diagnostic criteria for eosinophilic esophagitis: AGREE conference. Ann Allergy Asthma Immunol.2018;121:281-284.2. Spergel JM, Brown-Whitehorn TA, Muir A, Liacouras CA. Medical algorithm: Diagnosis and treatment of eosinophilic esophagitis in children. Allergy. 2020;75:1522-1524.3. Nhu QM, Aceves SS. Medical and dietary management of eosinophilic esophagitis. Ann Allergy Asthma Immunol.2018;121:156-161.4. Kagalwalla AF, Amsden K, Shah A, et al. Cow’s milk elimination: a novel dietary approach to treat eosinophilic esophagitis.J Pediatr Gastroenterol Nutr. 2012;55:711-716.5. Spergel JM, Elci OU, Muir AB, et al. Efficacy of Epicutaneous Immunotherapy in Children With Milk-Induced Eosinophilic Esophagitis. Clin Gastroenterol Hepatol. 2020;18:328-336 e327.6. Dioszeghy V, Mondoulet L, Dhelft V, et al. The regulatory T cells induction by epicutaneous immunotherapy is sustained and mediates long-term protection from eosinophilic disorders in peanut-sensitized mice. Clin Exp Allergy. 2014;44:867-881.Sincerely,Jonathan M. Spergel, MD, PhD1,2; Amanda B. Muir, MD2,3; Chris A. Liacouras, MD2,3;Deirdre Burke1, CRA; Megan O. Lewis, MSN, RN, CPNP1; Terri Brown-Whitehorn, MD1,2, Antonella Cianferoni, MD, PhD1,21Division of Allergy and Immunology, The Children’s Hospital of Philadelphia, PA, USA, 2Department of Pediatrics, The Perelman School of Medicine at University of Pennsylvania, Philadelphia, PA, USA, 3Division of Gastroenterology, Hepatology, and Nutrition, The Children’s Hospital of Philadelphia, PA, USAFunding Sources: The Children’s Hospital of Philadelphia Eosinophilic Esophagitis Family FundAcknowledgements: JMS, CAL, TBW and MOL are consultants for DBV Technology.Correspondence: Jonathan M. Spergel MD, PhDThe Children’s Hospital of PhiladelphiaDivision of Allergy and ImmunologyWood Bldg 3352D3401 Civic Center Blvd,Philadelphia, PA 19104Email: [email protected]: 1-215-590-2549Table 1

In addition to known allergens, other proteins in pollen can aid the development of an immune response in allergic individuals. The contribution of the “unknown” protein allergens is apparent in phylogenetically related species where, despite of high homology of the lead allergens, the degree of allergenic potential can vary greatly. The aim of this study was to identify other potentially allergenic proteins in pollen of three common and highly related allergenic tree species: birch (Betula pendula), hazel (Corylus avellana) and alder (Alnus glutinosa). For that purpose, we carried out a comprehensive, comparative proteomic screening of the pollen from the three species. In order to maximize protein recovery and coverage, different protein extraction and isolation strategies during sample preparation were employed. As a result, we report 2500 - 3000 identified proteins per each of the pollen species. Identified proteins were further used for a number of annotation steps, providing insight into differential distribution of peptidases, peptidase inhibitors and other potential allergenic proteins across the three species. Moreover, we carried out functional enrichment analyses that, interestingly, corroborated high species similarity in spite of their relatively distinct protein profiles. We provide to our knowledge first insight into proteomes of two very important allergenic pollen types, hazel and alder, where not even transcriptomics data is available, and compared them to birch. Datasets from this study can be readily used as protein databases, and as such serve as basis for further functional studies.

I thank Dres. Alnaes and Helnes Bergen for their stimulating comment on my medical algorithm on the Diagnosis and Treatment of Radiocontrast Media Hypersensitivity.1 In their comment, they raised attention to the possible addition of desensitization to radiocontrast media (RCM) management, which was not depicted in the algorithm.2 I have been well aware of several reports on desensitization and have already discussed them in a previous paper, however commented there that “successful desensitization of RCM has been reported for immediate hypersensitivity reactions to RCM, but it is only used anectodically” and concluded not to include this procedure into the algorithm.3In addition to the two papers on desensitization to RCM cited by Dr. Alaes, also a handful other cases have been published, some of them older. To my knowledge, at least as far as I can access these case reports, in none of these patients a proper allergy diagnosis and management has been performed and in most, if not all of these patients, desensitization probably was unnecessary. In the described cases, skin testing has not been performed or was even negative indicating a higher probability for a non-allergic immediate hypersensitivity reaction (IHR), in the history before desensitization was performed in several cases the RCM was not changed, but the same not tolerated RCM was given again and radiologists in vain relied on premedication to prevent recurrent attacks, and no skin test-negative RCM was identified and used. None of the cases published convinced me of the need for desensitization. Performing the examination with a skin test-negative RCM would with a high probability be successful.4 I would expect the success of desensitization was rather due to changing to a different isoosmolar RCM (and probably not to adding premedication) than the desensitization procedure itself, as alone changing the implicated RCM to another one in one study reduced the risk of recurrent IHR by 67.1% (odds ratio: 0.329; P = 0.001), whereas steroid premedication did not show protective effects.5Our group of European Network on Drug Allergy experts have highlighted that rapid desensitization is a procedure that can be used to provide a temporary tolerance to a first-line drug when no alternative is available.6 This implies for RCM hypersensitivity that using a skin-test-negative RCM for the next examination as an alternative drug is next step and not immediate desensitization. One problem with desensitization is that too many doctors employ it uncritically and without prior proper allergy workup, best with drug provocation test. The high rate of successful desensitizations without prior confirmation of drug hypersensitivity in the literature is in part explained by the fact that many of those patients would not have reacted anyway. I have yet to find convincing evidence to add desensitization as a standard therapeutic option to the RCM management algorithm.Having said this, I am eagerly following up the literature on RCM desensitization with great interest to be prepared, should I encounter an own patient, who would react severely to an alternative skin test-negative RCM after following the algorithm. Until now, colleagues and I have not met such a patient, however, I would seriously consider desensitization as an option in such a situation. Thus, I thank Dres. Alnaes and Helsen Bergen for bringing up that interesting topic for discussion.

In this review, we discuss recent publications on asthma and review the studies that have reported on the different aspects of the prevalence, risk factors and prevention, mechanisms, diagnosis and treatment of asthma. Many risk and protective factors and molecular mechanisms are involved in the development of asthma. Emerging concepts and challenges in implementing the exposome paradigm and its application in allergic diseases and asthma are reviewed, including genetic and epigenetic factors, microbial dysbiosis and environmental exposure, particularly to indoor and outdoor substances. The most relevant experimental studies further advancing the understanding of molecular and immune mechanisms with potential new targets for the development of therapeutics are discussed. A reliable diagnosis of asthma, disease endotyping and monitoring its severity are of great importance in the management of asthma. Correct evaluation and management of asthma comorbidity/multimorbidity, including interaction with asthma phenotypes and its value for the precision medicine approach and validation of predictive biomarkers are further detailed. Novel approaches and strategies in asthma treatment linked to mechanisms and endotypes of asthma, particularly biologicals, are critically appraised. Finally, due to the recent pandemics and its impact on patient management, we discuss the challenges, relationships, and molecular mechanisms between asthma, allergies, SARS-CoV-2 and Covid-19.

In the newly published article: “Medical algorithm: Diagnosis and treatment of radiocontrast media hypersensitivity” K. Brockow [1], suggested an algorithm for the evaluation of hypersensitivity to radiocontrast media (RCM). However, one important aspect has been left out of the suggested algorithm, the opportunity to desensitize patients with probable or confirmed immediate hypersensitivity reactions to radiocontrast media.  Especially those reacting to more than one substance. Desensitization to allergens is a standard preventative measure of anaphylaxis when patients have a history of severe immediate hypersensitivity reactions to a drug, and there are no therapeutic alternatives [2]. Protocols for RCM desensitization are known from case reports [3,4], and need more work and research before standardized protocols can be recommended, but they can facilitate the use of radiocontrast media in important situations such as the need for acute coronary angiograms. Such situations are rare, but an algorithm should include information and pathways to facilitate the best treatment possible for all patients. Suggested change in the algorithm is to include the option “rapid desensitization” in the pathway “RCM urgently needed without test possibility and indispensable need for the RCM”, and in the pathway for ”patients with confirmed RCM hypersensitivity with immediate hypersensitivity reactions to several RCM’s for situations with indispensable need for RCM”. This would help to facilitate the best point of care for more patients with hypersensitivity to RCM. Dr. Alnæs has nothing to disclose. Dr Alnæs is the only contributor to this article.  1.     Knut Brockow, Medical algorithm: Diagnosis and treatment of radiocontrast media hypersensitivity Allergy 2020 ;75:1278-1280. doi: 10.1111/all.14147. 2.     P. Demoly  N. F. Adkinson  K. Brockow et al. International Consensus on drug allergy, Allergy 2014; 69: 420-437.  https://doi.org/10.1111/all.123503.     Mona Al-Ahmada and Tito Rodriguez Bouzab ,Successful desensitization to radiocontrast media in two high-risk cardiac patients. Ann Saudi Med. 2017; 37(4): 333–335. doi: 10.5144/0256-4947.2017.333 4.     Saurav Uppal , Anthony E DeCicco , Anselma Intini  et al. Rapid Desensitization to Overcome Contrast Allergy Prior to Urgent Coronary Angiography. Int Heart J  2018; 30; 59(3):622-625. doi: 10.1536/ihj.17-395.

A document by Asami Fukuda. Click on the document to view its contents.

Trained immunity refers to the fact that the innate immune system also demonstrates memory, resulting in a faster and more profound second innate reaction, days to weeks after a first reaction to another pathogen or vaccine. Thus, trained immunity is heterologous, non-specific. We applied this principle with MMR vaccination during the COVID-19 pandemic.In a prospective, observational, single-center study 255 subjects, most at high risk for infection with COVID-19, received preventive MMR vaccination; 36 got infected with COVID-19; all had a mild course, even though 40% had risk factors. This might in part be due to trained immunity, conveying innate immune memory secondary to MMR vaccination, enhancing the innate immune response once the subject gets infected with SARS-CoV-2.As a result the well-known immune suppression brought about by coronavirus might not work so well, as the innate immune system is primed, allowing the body to finally eliminate the virus more efficiently.

Background: Peanut allergy has a rising prevalence in high-income countries, affecting 0.5–1.4% of children. This study aimed to better understand peanut anaphylaxis in comparison to anaphylaxis to other food triggers in European children and adolescents. Methods: Data was sourced from the European Anaphylaxis Registry via an online questionnaire, after in-depth review of food induced anaphylaxis cases in a tertiary paediatric allergy centre. Results: 3514 cases of food anaphylaxis were reported between July 2007 - March 2018, 56% in patients younger than 18 years. Peanut anaphylaxis was recorded in 459 children and adolescents (85% of all peanut anaphylaxis cases). Previous reactions (42% vs 38%; p=0.001), asthma comorbidity (47% vs 35%; p<0.001), relevant co-factors (29% vs 22%; p=0.004) and biphasic reactions (10% vs 4%; p=0.001) were more commonly reported in peanut anaphylaxis. Most cases were labelled as severe anaphylaxis (Ring&Messmer grade III 65% vs 56% and grade IV 1.1% vs 0.9%; p=0.001). Self-administration of intramuscular adrenaline was low (17% vs 15%), professional adrenaline administration was higher in non-peanut food anaphylaxis (34% vs 26%; p=0.003). Hospitalisation was higher for peanut anaphylaxis (67% vs 54%; p=0.004). Conclusions: The European Anaphylaxis Registry data confirmed peanut as one of the major causes of severe, potentially life-threatening allergic reactions in European children, with some characteristic features e.g. presence of asthma comorbidity and increased rate of biphasic reactions. Usage of intramuscular adrenaline as first line treatment is low and needs to be improved. The Registry, designed as the largest database on anaphylaxis, allows continuous assessment of this condition.

To the Editor: Asthma is a complex and heterogeneous chronic airway inflammatory disease with the involvement of environmental factors through epigenetic mechanisms.1 Accordingly, repeated injury, repair and regeneration of the airway epithelium following exposure to environmental factors and inflammation results in histological changes and functional abnormalities in the airway mucosal epithelium, which are associated with the pathophysiology of asthma.2Epigenetics is defined by heritable changes in gene expression without changes in the DNA sequence.3 Regulation of gene expression is mediated by different mechanisms such as DNA methylation, histone modifications and RNA-associated silencing by small non-coding RNAs. CpG sites are dinucleotides consisting of guanine and cytosine concentrated in clusters referred to CpG islands found at important regulatory sites, such as promoter and enhancer regions.4 Their de novo methylation occurs in response to various cellular stressors and signals by DNA methyltransferases (DNMT3a and 3b), which add a methyl group to position 5 of cytosine residues at the CpG site. During DNA replication both of the separated strands of DNA carry one methylated cytosine to be used as a template for duplication. Daughter DNA duplex strands will thus be hemi-methylated, which is recognized by a different DNA methyltransferase isoform (DNMT1).5 Because DNA methylation is a reversible process, the DNMTs are considered as a therapeutic target. Several DNMT inhibitors have been identified recently, among the non-nucleoside inhibitors, 4-aminoquoline-based inhibitors, such as SGI-1027 showed potent inhibitory activity. SGI-1027 occupies the binding site of DNMTs resulting in the prevention of access of target DNA to the substrate binding pocket.6We have demonstrated in previous studies from our laboratory that human primary bronchial epithelial cells (HBEC) isolated from patients with asthma showed lower barrier integrity compared to controls.7 To investigate the level of global methylation in HBEC, we investigated control and asthma samples for the long interspersed nuclear element-1 (LINE-1) methylation levels (Figure 1A). HBEC from asthma patients showed a tendency for higher global methylation levels, together with higher expression of 5-methylcytosine (5-mc) in immunofluorescence staining (Figure 1B). Next, we performed methylation profiling (Illumina Infinium EPIC array) to investigate genes methylated in ALI cultures of HBEC. Interestingly, in a highly methylated group of top 100 genes, we found many genes associated with cell growth, ion transport, and cytoskeletal remodeling (Figure S1). We kept our attention on the methylated epigenetic and tight junction (TJ) genes and further focused on TJs, especially zonula occludens and claudins which showed higher methylation in contrast to occludin, which was not methylated (Figure S2). As higher methylation levels were observed in HBEC of asthmatic origin, we inhibited the DNA methyltransferase enzyme with a specific inhibitor, SGI-1027, to demonstrate the role of CpG methylation on epithelial barrier integrity. ALI cultures were treated with the DNA methyltransferase inhibitor for 72 hours. Significantly decreased expression of 5-mc was observed after 48 hours of DNA-methyltransferase inhibition, demonstrating that the methylation of 5-methylcytosine (5-mc) in bronchial epithelium was reversed (Figure 2A). This prompted us to investigate the changes triggered by the inhibitor in epithelial cells. Further experiments showed increased transepithelial electric resistance (TER) in bronchial epithelial cells, in ALI from asthmatic donors after 48 hours of DNMT inhibition (Figure 2B). The link between barrier integrity and TER results were confirmed by the significantly decreased paracellular passage of FITC-labelled 4kD dextran after inhibition of DNMTs (Figure 2C). The reconstitution of TER in asthmatic ALI was associated with decreased protein DNMT1 expression and increased ZO-1 and claudin-18 proteins (Figure 2D). We also observed increased claudin-4, but not occludin expression upon DNMT inhibition (Figure S3). Increased expression of ZO-1 with an intact and honeycomb-like structure in the immunofluorescence staining of bronchial epithelial cells confirmed the effect on protein expression of bronchial epithelial barrier in asthma donors (Figure S4).Defective epithelial barrier has been established in asthma in addition to several chronic inflammatory diseases.8 Direct targeting of the epithelial barrier leakiness for the treatments represents an important target, however so far there is no treatment possibility targeting epigenetic mechanisms. The present study demonstrates an increased global methylation level in HBEC from asthmatic individuals. CpG methylation of specific genes is essential for the defect of epithelial barrier integrity, which is reversed upon DNMT inhibition. The inversion of CpG methylation, restores leakiness in the epithelium in asthma by increasing TER, decreasing paracellular flux and improves the structure of bronchial epithelial cells by increasing the expression of TJ proteins. The better understanding of the importance of epigenetic memory in chronic tissue inflammatory diseases together with the availability of treatment modalities targeting epigenetic mechanisms and transition of these molecules into the clinical studies may lead to curative treatment of allergic and autoimmune inflammatory diseases.9Paulina Wawrzyniak1, PhD,Krzysztof Krawczyk1,3, MSc,Swati Acharya5, PhD,Ge Tan1,7, PhD,Marcin Wawrzyniak1, PhD,Emmanuel Karouzakis4, PhD,Anita Dreher, Sci. Tech.,Bogdan Jakiela2, MD, PhD,Can Altunbulakli1, PhD,Marek Sanak2, MD, PhD,Liam O‘Mahony1,6, PD, PhD,Kari Nadeau5, MD, PhD,Cezmi A. Akdis1, MD1Swiss Institute of Allergy and Asthma Research (SIAF), University of Zürich, Davos, Switzerland, Christine Kühne-Center for Allergy Research and Education (CK-CARE)2Department of Medicine, Jagiellonian University Medical College, Krakow, Poland3Faculty of Biology and Environmental Protection, Department of Cellular Immunology, Lodz, Poland4Department of Rheumatology, University Hospital of Zurich5Departament of Medicine, Stanford University, United States6 Department of Medicine and School of Microbiology, APC Microbiome Ireland, University College Cork, Cork, Ireland.7 Functional Genomics Center Zurich, ETH Zurich/University of ZurichCorresponding author:Paulina WawrzyniakSwiss Institute of Allergy and Asthma Research (SIAF), University of Zürich, Davos, SwitzerlandObere Strasse 22,7270 Davos, SwitzerlandTel: +41 81 410 08 48Fax: +41 81 410 08 [email protected] of interest:The authors declare that they have no conflicts of interest.Founding sources:Supported by Swiss National Science Foundation grants 310030_156823, and 320030_176190.Word count: 765Keywords: asthma, tight junction, CpG methylation, DNA methyltransferases,

To the Editor: The spread of the coronavirus disease-2019 (COVID-19) remains a worsening global health crisis. Although many studies have reported risk factors for severe COVID-19, asthma characterization in COVID-19 is still controversial, with different early reports from China and recent reports from the Europe and United States.1 Prolonged viral shedding is not only a risk factor for poor outcome of COVID-19, but also clues to host immune response against the virus. However, there is limited data on this except for results from relative small group studies.2 In this study, 2 200 adult patients hospitalized for COVID-19 in Daegu were evaluated for prevalence of asthma and clinical outcomes with COVID-19 according to asthma. In addition, the risk factors for delayed viral clearance were evaluated.The prevalence of asthma in patients with COVID-19 was 3.2% which was not different from its prevalence in the Korea National Health and Nutrition Examination Survey (KNHANES) (Figure 1A and Table S1). By age group, the prevalence of asthma showed a similar U-shaped pattern as the general prevalence pattern in Korea. However, the prevalence of asthma in the 19–29-year age group (2.1%) was lower than that of KNHANES (Figure 1B).Table S2 compares the characteristics between the asthma group and the non-asthma group. Older age, overweight, and comorbidity of chronic obstructive pulmonary disease, and initial symptoms of dyspnea and nausea/vomiting were more common in the asthma group. Compared with the non-asthma group, the asthma group had a greater risk of death (13.6%vs. 6.4%, P = 0.02) and a greater need for high-flow oxygen therapy (18.2% vs . 10.5%, P = 0.048) (Figure 1C and Table S3). The higher mortality rate in asthma patients compared with non-asthmatic patients was particularly noticeable in female and overweight patients. Older patients (> 65 years) with asthma tended to have a higher mortality rate than those without asthma (Figure 1D). After adjusting for potential confounders, asthma had no significant association with clinical outcomes of COVID-19 (Figure 1E and Table S4). Meanwhile, older age, male gender, and comorbid diseases including overweight, diabetes, chronic kidney disease, cancer, autoimmune disease, dementia, and other psychological disorder were significant risk factors for mortality (Tables S5 and S6).Asthma is considered to have a lower risk of death than other well-known risk factors.3, 4 However, asthma is a heterogeneous disease and is often associated with atopic and eosinophilic asthma in younger patients. Meanwhile, obese asthma and elderly asthma are known to have common neutrophilic phenotypes.5, 6 The recent results of higher expression of COVID-19 receptors in respiratory specimens with neutrophilic asthma phenotype compared with the eosinophilic asthma phenotype.7 Considering prevalence and clinical outcome results, it is possible that neutrophilic asthma is a risk factor for infection and poor prognosis of COVID-19 rather than eosinophilic asthma.When delayed viral clearance was divided into two groups based on 30 days, 906 patients were included in the non-delayed viral clearance group and 415 patients in the delayed viral clearance group. After adjusting for potential confounders, delayed viral clearance was not significantly associated with asthma (Figure 1E and Table S4). However, older age >65 years (Odds ratio (OR) 2.002, 95% Confidence interval (CI) 1.292–3.101; P = 0.002), comorbid diseases including dementia (OR 3.123, 95% CI 1.833–5.321; P<0.001), and other psychological disorder (OR 2.084, 95% CI 1.178–3.687; P = 0.012), initial symptom of skin rash (OR 15.943, 95% CI 1.613–157.535; P = 0.018), and initial laboratory abnormalities including hemoglobin <10 g/dL (OR 2.156, 95% CI 1.161–4.003; P = 0.015) and C-reactive protein (CRP) ≥1.0 mg/dL (OR 1.588, 95% CI 1.061–2.377; P = 0.025) were significant risk factors for delayed viral clearance. On the other hand, male sex (OR 0.752, 95% CI 0.567–0.997; P = 0.047), hypertension (OR 0.704, 95% CI 0.519–0.953; P = 0.023), and initial symptom of headache (OR 0.673, 95% CI 0.485–0.932; P = 0.017) were significant protective factors for delayed viral clearance (Figure 2A and Table S7). In particular, when limited to the mild COVID-19 group classified as no activity limitations in the outcome parameters, older age, dementia, initial symptoms of skin rash and headache, and initial hemoglobin <10 g/dL showed significant differences (Figure 2B, Table S8).Several factors related to the nervous system were identified as important risk factors for delayed viral clearance. Previous studies have shown that the coronavirus can initially invade the peripheral nerves and enter the central nervous system through a synapse path.8 It is hypothesized that the ability of the immune system to find and remove viruses that have penetrated the nervous system is important for virus clearance. Male sex, hypertension and elevated CRP did not show a significant difference when analyzed only mild patients, and these may be indicators associated with severity rather than a direct effect on viral clearance.Anti-inflammatory drugs such as hydroxychloroquine and systemic steroid were shown to be risk factors for mortality and delayed viral clearance (Table S6 and S8). These medications were used more often when the hospitalization period was extended or when showing poor prognosis factors. Notwithstanding these, our results suggest that anti-inflammatory drugs need to be used with proper consideration of appropriate indications.On May 9, 2020, there were 6,859 patients with PCR-confirmed COVID-19 in Daegu. This data excluded asymptomatic or minimal symptomatic patients who did not require hospitalization. However, our study covered almost all hospitalized patients diagnosed with COVID-19 in Daegu from February to May ,therefore, selection bias is minimized.9 In Korea, most hospitals decided to terminate the quarantine by repeating PCR every week. In addition, the Korea Centers for Disease Control & Prevention (KCDC) thoroughly managed the criteria for quarantine termination and PCR results. Through this, in our study, we were able to perform a large-scale study to confirm the risk factors for delayed viral clearance.In summary, despite the positivity of differences depending on phenotypes, the prevalence of asthma was not significantly different in patients with COVID-19, and asthma did not affect the outcomes of COVID-19. Age, dementia, and initial presentations of headache, skin rash, and anemia were independently associated with viral clearance.

Drug hypersensitivity reactions (DHRs) represent a global threat to healthcare systems due to their incidence, with a significant increase over last years1. DHR figures are overestimated in the general population since less than 40% of cases initially labelled as allergic can be confirmed as such when evaluated in an allergy unit2. Achieving an accurate diagnosis is complex and time consuming; besides, tests must be tailored to specific clinical manifestations and underlying mechanisms and will depend on the culprit drugs. Diagnosis often requires performing drug provocation tests (DPTs), which are especially problematic for severe reactions, making management of these patients challenging and expensive for the health care system.Clinically, DHRs are classified into immediate and non-immediate, based on the time interval between drug exposure and onset of the symptoms3. The most severe immediate reaction is anaphylaxis. This issue of the journal has been dedicated o drug hypersensitivity, which is becoming a major public health issue during the last decade, especially with the introduction of biologicals to medicine. Bilo et al. 4 evaluated the anaphylaxis mortality rate in Italy from 2004 to 2016 and found an average mortality rate for definite anaphylaxis (ICD-10 code) of 0.51 per million population per year, mostly due to the use of medications (73.7%), although in 98% of the cases culprit drugs were not identified. Regarding non-immediate reactions, one of the most challenging diagnoses is drug reaction with eosinophilia and systemic symptoms (DRESS), which is sometimes difficult, at an early stage, due to overlapping clinical symptoms with maculopapular exanthema (MPE). Pedruzzi et al. 5 identified 7 microRNAs (miRNAs) that correctly classified DRESS or MPE patients and were associated with keratinocyte differentiation/skin inflammation, type I IFN pathway viral replication, ATP-binding cassette transporters, and T lymphocyte polarisation, being all of them potential biomarkers. Non-immunologically mediated adverse reactions, such as attention-deficit/hyperactivity disorder (ADHD) are reported by Fuhrmannet al. 6 in association with systemic H1-antihistamines administration in school-age children, especially the 1st generation agents.The mechanism underlying DHR and the reason why patients treated with the same drug develop a tolerance response or an immediate or non-immediate DHR is not completely understood (Figure 1). Therefore, the prediction of who may experience a DHR, and if so, in what form, remains clinically obscure for most drugs. Goh SJR et al. 7 elegantly analyse this complexity, using non-immediate reactions to penicillins as a model. They focus on the understanding of the role of nature of the lesional T cells, the characterisation of drug-responsive T cells isolated from patient blood, and the potential mechanisms by which penicillins enter the antigen-processing and presentation pathway to stimulate these deleterious responses.Regarding specific drugs involved in allergy, betalactam antibiotics (BL) are the most frequent culprit, being many reactions mediated by IgE. This type of reaction varies among patients, with some reacting only to one BL and others to several of them; it tends to change over time and differs between European countries, depending on BL consumption. Nowadays, amoxicillin (AX), alone or in combination with the β-lactamase inhibitor clavulanic acid (CLV), is the most often prescribed BL worldwide (Figure 2) and the most common elicitor of reactions in both children and adults. It is unclear why patients after the administration of AX-CLV develop selective hypersensitivity to AX, while tolerating CLV and vice-versa. Ariza et al. 8 generated drug-specific T-cell clones from AX- or CLV-selective immediate hypersensitivity patients and found that both AX- and CLV-specific clones were generated irrespective of whether AX or CLV was the culprit, although a higher secretion of Th2 cytokines (IL-13 and IL-5) was detected when clones were activated with the culprit BL compared with clones stimulated with the tolerated BL, in which higher secretion of Th1 cytokines (IFN-γ) was observed. Regarding selective non-immediate reactions to CLV, Copaescu A et al. 9 report on a cohort of patients with a history of non-immediate reaction to CLV, who demonstrated a delayed intradermal skin test response to CLV, 17% were allergic to both CLV and ampicillin, and 83% were selective reactors with good tolerance to AX. IFN-γ release enzyme-linked immunospot (ELISpot) was performed giving a sensitivity of 33%. Other drugs such as sulphonamides, either antibiotic or non-antibiotics are important triggers of non-immediate DHRs. Vilchez-Sanchez et al. 10 showed that lymphocyte transformation tests (LTT) can help avoid the performance of DPT with a sensitivity of 75%, a specificity of 100%, and negative and positive predictive values of 72.7% and 100%, respectively.There has been a great expansion in the use of biological agents (mainly monoclonal antibodies (mAbs)), and they have greatly improved the treatment landscape of hemato-oncologic, autoimmune, inflammatory and rheumatologic diseases. In parallel, the incidence rate of reported DHRs associated with these products has increased considerably within the last years, ranging from mild to life-threatening. Yang BC et al. 11 recommend risk stratification as the first step for managing patients with DHRs to these drugs. In cases with negative skin test and mild reactions, DPT is an option, and in moderate or severe reactions, desensitisation becomes the preferred approach. In cases with positive skin test, desensitisation is the recommended course of action, especially when there is no alternative medication. Desensitisation is also widely used in the management of immediate hypersensitivity reactions to chemotherapy agents, such as platinums. There is suspicion about the presence of a longer memory of tolerance in subsequent desensitisation protocols partially resembling the regulatory tolerance mechanisms induced by allergen immunotherapy. Tüzer et al. 12 demonstrate the possible role of IL-10 in desensitisation with platinums, as they found a dynamic change in serum IL-10 levels observed as an increase during desensitisation and a decrease in between the protocols.Finally, a wide spectrum of drugs has been considered for treatment of coronavirus disease 2019 (COVID-19) and all of them can potentially induce DHRs. Gelincik A et al .13 reviewed DHRs in COVID-19 times to these drugs, with knowledge mainly coming from previous clinical experience in patients not infected with COVID-19. As in other viral infections, skin symptoms, including exanthemas, may appear during the evolution of the disease, leading to differential diagnosis with DHRs. Whether COVID-19 can aggravate T–cell mediated DHRs reactions as some viruses is at present unknown.We can conclude that new drugs are continuously introduced into the markets and confirmed as inducers of hypersensitivity reactions. We still do not completely understand the mechanisms underlying many of these reactions and further studies for improving diagnostic and management are needed even in classic and well-studied drugs as BLs.Abbreviations: AX: Amoxicillin; CLV: Clavulanic acid; COVID-19: Coronavirus disease 2019; DHR: Drug hypersensitivity reactions; DPT: Drug provocation tests; DRESS: Drug reaction with eosinophilia and systemic symptoms; ELISpot: enzyme-linked immunospot; LTT: Lymphocyte transformation tests; MPE: Maculopapular exanthema.