Predicting reaction threshold and severity are important to improve the management of food allergy, however the determinants of, and relationship between, these parameters are significant knowledge gaps. Identifying robust predictors could enable the reliable risk-stratification of food-allergic individuals. In this series of young people with CM-allergy undergoing DBPCFC – the largest reported in the literature – we did identify any baseline marker which predicted the occurrence of anaphylaxis at challenge, consistent with existing data. 1 There is one report of IgE-sensitisation being predictive of severity in CM-allergy, 5 however the authors included non-reactive patients in their analysis which significantly skewed the analyses, resulting in misleading conclusions. 6 IgE-sensitisation in our cohort, particularly to casein, was predictive of LOAEL. Including an assessment of casein IgE may therefore be of clinical utility when evaluating patients with CM-allergy in the clinical setting.

Most patients presenting with allergies are first seen by primary care health professionals. The perceived knowledge gaps and educational needs were recently assessed in response to which the LOGOGRAM Task Force was established with the remit of constructing pragmatic flow-diagrams for common allergic conditions in line with an earlier EAACI proposal to develop simplified pathways for the diagnosis and management of allergic diseases in primary care. To address the lack of accessible and pragmatic guidance, we designed flow-diagrams for five major clinical allergy conditions: asthma, anaphylaxis, food allergy, drug allergy and urticaria. Existing established allergy guidelines were collected and iteratively distilled to produce five pragmatic and accessible tools to aid diagnosis and management of these common allergic problems. Ultimately, they should now be validated prospectively in primary care settings.

Background It has been hypothesized that epigenomic modifications such as genomic methylation changes are an intermediate step linking environmental exposures with allergic disease development. Associations between individual DNA methylation CpG sites and allergic diseases have been reported, but they have not been assessed regarding their joint predictive capability. Methods Data were obtained from 240 children of the German LISA cohort. Blood-based DNA methylation was measured at six and ten years. Aeroallergen sensitization, at least RAST class 1, was measured in blood at six, ten and 15 years. We calculated six methylation risk scores (MRS) for allergy-related phenotypes based on available publications and assessed their performance both cross-sectionally and prospectively. Dose-response associations between aeroallergen sensitization and MRS, their correlation and mapping of common hits were evaluated. Results All six atopy-related MRS were highly correlated (r>0.86) and seven CpGs were included in more than one MRS. Cross-sectionally, we observed an 80% increased risk for aeroallergen sensitization at six years with an increased risk score by one standard deviation (best MRS: relative risk = 1.81, 95% confidence interval = [1.43; 2.27]). Significant associations were also seen at ten years and in prospective models, though the effect of the latter was attenuated when only including participants not sensitized at baseline. A clear dose-response relationship with RAST classes of aeroallergen sensitization could be established cross-sectionally, but not prospectively. Conclusion We found good classification and prediction capabilities of calculated allergy-related MRS, particularly cross-sectionally for the allergy prevalence, underlining the relevance of altered gene-regulation in allergic diseases.

World Health Organization Global Air Quality Guideline Recommendations: Executive SummaryAnna Goshua1, Cezmi Akdis2, Kari C. Nadeau3,4 1Stanford School of Medicine, Stanford, CA, USA2Swiss Institute of Allergy and Asthma Research (SIAF), University Zurich, Davos, Switzerland3Sean N. Parker Center for Allergy and Asthma Research at Stanford University, Stanford, CA, USA4Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, Stanford University, Stanford, CA, USACorresponding Author: Kari C. Nadeau, Sean N. Parker Center for Allergy and Asthma Research at Stanford University, Stanford University, Stanford, CA, USA; Email: [email protected] of Interest: Dr Cezmi Akdis reports research grants from Allergopharma, Idorsia, Swiss National Science Foundation, Christine Kühne-Center for Allergy Research and Education, European Commission’s Horison’s 2020 Framework Programme “Cure”, Novartis Research Institutes, Astra Zeneca, research grants and advisory board from Glaxo Smith-Kline, Sanofi/Regeneron, Scibase, Novartis, and is Editor-in-Chief of Allergy. All other authors declare no conflict of interest.Text Word Count: 1243Abstract Word Count: 147Figure/Table Count: 2Reference Count: 9

There is increasing understanding, globally, that climate change and increased pollution will have a profound and mostly harmful effect on human health. This review brings together international experts to describe both the direct (such as heat waves) and indirect (such as vector-borne disease incidence) impacts of climate change depending on their vulnerability (i.e., diseases) on an international, economic, political and environmental context. This unique review also expands on these issues to address a third category of potential longer-term impacts on global health: famine, population dislocation, and environmental justice and education. This scholarly resource explores these issues fully, linking them to global health in urban and rural settings in developed and developing countries. The review finishes with a practical discussion of action that health professionals around the world in our field can yet take.

This is a “Letter to the Editor” with no abstract.

Initial guidelines advised that sensitization to PEG should be taken into consideration in suspected subjects before a recommendation on the administration of vaccines for COVID-19 containing PEG or its cross-reactive analogues. 3 However, PEG shows an important variability in terms of molecular weights and conjugation forms. In that sense, although it is known that PEG-2000 (MW: 2000g/mol) conjugated with lipids is the form contained in the vaccines for COVID-19, there has been great variability in the PEG molecules used in allergy tests to evaluate sensitization of suspected subjects in the context of the current COVID-19 vaccination campaigns. 4 In this context, recent findings have shed light on the specific form of PEG that could be responsible of the hypersensitivity reactions to the mRNA vaccines for COVID-19.

Addressing Beta-lactam Allergy: A Time for actionElizabeth J. Phillips, MD, FIDSA, FAAAAI, Pascal Demoly, MD, PhD, Maria J Torres, MD, PhD1 Department of Medicine, Center for Drug Safety and Immunology, Vanderbilt University Medical Center, Nashville Tennessee USA, 2Institute for Immunology & Infectious Diseases, Murdoch University, Murdoch Australia, 3Division of Allergy, Department of Pulmonology, University Hospital of Montepellier, and IDESP, Univ. Montpellier – Inserm, Montpellier France,4Allergy Unit, Hospital Regional Universitario de Malaga-IBIMA-BIONAND-ARADyAL, and Departmento de Medicina, Universidad de Malaga, Malaga, SpainCorrespondence:Elizabeth J. Phillips, MD, FIDSA, FAAAAICenter for Drug Safety and ImmunologyVanderbilt University Medical Center1161 – 21st Avenue SouthNashville, TN 37232(615) 322-9174 (tel)[email protected] is now 93 years since the discovery of penicillins, and over 75 years since the first use of penicillin. We have entered yet another wave of challenges plagued with antibiotic resistance accelerating at a rate that well exceeds that of new antibiotic development. In the face of these uphill battles, 8-15% of a global population who has had access to care is labeled as penicillin allergic.1 In the United States (US) there are at maximum 6000 specialists who practice allergy out of a total of 700,000 practicing physicians, and not all allergists are proficient in and practice drug allergy. Conservatively out of 30,000,000 who are labeled as penicillin allergic at any one time in the US, this would mean that each allergist would need to delabel a minimum of 6000 patients. In Europe and the United Kingdom, the figures are proportionately identical, with some differences between countries. Even if all patients had equal access to care, this type of scalability remains impossible. This overwhelming burden that threatens to negatively impact healthcare through delays in treatment, higher healthcare utilization and cost, less effective treatment and increased antibiotic resistance and Clostridioides difficile infection, demands a risk-based approach that simplifies the penicillin allergy delabeling process and establishes bridges with non-allergists.1, 2What have we learned that now makes the population level goal of penicillin delabeling achievable? First off, prevention is better than cure. We should critically examine pediatric populations for antibiotic use to address over-prescription of antibiotics including penicillins for viral infections. We should avoid labeling children with benign delayed exanthems that occur in the setting of a likely viral infection as penicillin allergic. When continued treatment is necessary we should in fact encourage “treating through” such reactions. When a label of penicillin allergy seems inevitable in a child we should address this label early and pay particular attention to antibiotic stewardship. New data on serum sickness-like reaction suggests that many of these are likely virally mediated and do not reproduce on ingestion challenge.3 Community based education programs can help disseminate timely information on penicillin allergy to dispel myths and alleviate fears. A label of penicillin allergy should be both viewed and approached as a threat to both individual and public health. On a public health level addressing penicillin allergy should be seen as a broad stewardship tool that provides a level of herd protectiveness against antibiotic resistance. On an individual level a label of penicillin allergy should be approached with the same routineness as any other preventative health check, and primary care physicians and providers should be trained to understand and manage low-risk penicillin allergy labels.4 Patients should regularly discuss their drug allergy passport with their healthcare providers such as pharmacists and physicians. Allergy passports should enable interoperability, high traceability and time-stamped information solving the problem of frequent unavailability and inaccuracy of drug allergy information.5 Risk stratification should occur and if in a low-risk category a patient should be given the option of direct oral challenge and delabeling. Risk stratification to identify by clinical history the low-risk penicillin allergic patients who would be appropriate for simple procedures is key. Several mechanisms now exist to risk stratify those labeled as penicillin allergic in routine clinical practice. These clinical prediction rules provide an evidence base to identify the majority of low-risk penicillin allergy labeled patients who are at low risk for rechallenge reactions.6, 7 In current practice it is likely that less than 1% of such low-risk patients will be at risk for a reaction on ingestion challenge.1, 8To make widespread penicillin allergy delabeling an achievable and scalable goal we must be convinced of the safety of direct ingestion challenges. A randomized study allocated children 5 years or older with low-risk cutaneous reaction to penicillin skin testing followed by amoxicillin challenge versus 2 step direct oral challenge with amoxicillin with tolerance of amoxicillin of 96% of those with direct challenge and only minor reactions in the remainder.9These results have recently been confirmed in an European population of children.10 Aside from the inconvenience and potential need for specialty assessment, for very low-risk patients, the use of skin testing would be expected to perform poorly considering their low pre-test probability of a reaction. Several other studies have demonstrated that a single or two-step direct ingestion challenge with penicillins such as amoxicillin is a safe and practical strategy to remove a label of penicillin allergy.11 Although there is evidence to support the use of risk stratification tools to delabel penicillin allergy under allergist guidance, we require an educational program on drug allergy for primary care physicians as well validation of these risk stratification tools, to show that low-risk penicillin delabeling can be achieved in this setting.Even in the face of risk stratification and safety of direct ingestion challenge, populations are not equal in terms of their medical risk or antibiotic needs. Intuitively populations that serve to benefit from penicillins and other beta lactams have been shown to have inferior outcomes when labeled as penicillin allergic that would benefit from a delabeling intervention. This includes the association of penicillin allergy label and use of an alternative antibiotic with post-operative surgical site infections.12 Other settings where research has shown feasibility in delabeling include children in the emergency department, critically ill populations with high antibiotic needs, and pregnant women where the high rates of surgical delivery and group B Streptococcal colonization in pregnancy create a high demand for penicillin and cephalosporins as safe firstline drugs.1, 13, 14 Increasingly, assessment of unverified penicillin allergy has been recognized as an antibiotic stewardship intervention in immunocompromised states such as transplant and cancer where populations have much to gain by being delabeled.15There is a “time for action” for removal of penicillin allergy labels on a population level but how do we achieve widespread implementation (Figure 1)? Policy changes should be driven by collaboration with Infectious diseases specialists and allergists who should join forces to pair antibiotic allergy management with antibiotic stewardship. In the community we need to educate parents and pediatricians to make them aware of the hazards of both unnecessary antibiotics and penicillin allergy labels for mild rashes that are often related to a viral infection and unlikely to recur. Primary healthcare providers should be given greater incentives to delabel penicillin allergic patients at the point-of-care and armed with decision support tools to facilitate risk stratification. For those whose history is not consistent with allergy this could include direct delabeling without testing. In the future, evidence may support that routine direct ingestion challenge with a penicillin and delabeling is safe in the primary care setting. Finally, by off-loading low-risk reactions to primary care providers we can then prioritize care of the patients with a higher-risk allergy and/or medical history by engagement with specialists who can provide more in-depth assessments and give them the best antibiotic options.Figure 1: Addressing Beta-lactam Allergy: An Implementation Roadmap: There are currently many missed opportunities for community members and healthcare providers to take action forward on the “penicillin allergy delabeling” movement. This includes not only active measures to delabel patients by history and direct oral challenge and to identify high risk patients for prioritized penicillin allergy delabeling but also preventive measures to avoid unnecessary use and exposure to antibiotics and avoidance of unnecessary labeling in those with mild rashes of likely viral origin.

A document by Laura Haya. Click on the document to view its contents.

Disrupted epithelial barrier in nasal polyps characterizes aspirin exacerbated respiratory disease Anand Kumar Andiappan1*, Mohammad Asad2, Celine Chua3, Esha Sehanobish2 , Zhen Ren4, Xue Ying Chan1, Josephine Lum1, Nicholas Ang1, Duan Kaibo1, Adam Gersten2, Waleed M. Abuzeid5, Nadeem Akbar2, Marc Gibber2, Shanshan Howland1, Bernett Lee1, Olaf Rotzschke1, Steven A. Porcelli2, Elina Jerschow2*1 Singapore Immunology Network, Agency for Science, Technology and Research2 Albert Einstein College of Medicine/Montefiore Medical Center, Bronx, NY, USA3 Department of Biological Science, National University of Singapore4 Division of Allergy and Immunology, Department of Medicine, Washington University Schoolf of Medicine, St Louis, MO, USA5Rhinology and Endoscopic Skull Base Surgery, Department of Otolaryngology – Head and Neck Surgery, University of Washington, Seattle, WA, USA*Correspondence: [email protected]; [email protected]

Background Limited information exists on nursing home (NH) residents regarding BNT162b2/Pfizer vaccine efficacy in preventing SARS-CoV-2 and severe Covid-19, and its association with post-vaccine humoral response. Methods 396 residents from seven NHs suffering a SARS-CoV-2 B.1.1.7 (VOC-α) outbreak at least 14 days after a vaccine campaign were repeatedly tested using SARS-CoV-2 real-time reverse-transcriptase polymerase chain reaction on nasopharyngeal swab test (RT-PCR). SARS-CoV-2 Receptor-Binding Domain (RBD) of the S1 subunit (RBD-IgG) was measured in all residents. Nucleocapsid antigenemia (N-Ag) was measured in RT-PCR-positive residents, and serum neutralizing antibodies in vaccinated residents from one NH. Results The incidence of positive RT-PCR was lower in residents vaccinated by two doses (22.7%) vs one dose (32.3%) or non-vaccinated residents (43.7%)(p<0.01). Covid-19-induced deaths were observed in 10.4% of the non-vaccinated residents, in 6.4% of those who had received one dose, and in 0.9% with two doses (p=0.0007). Severe symptoms were more common in infected non-vaccinated (21.0%) vs vaccinated residents (47.6%, p=0.002). Higher levels of RBD-IgG (n=325) were associated with a lower SARS-CoV-2 incidence. No in vitro serum neutralization activity was found for RBD-IgG levels below 1,050 AU/mL. RBD-IgG levels were inversely associated with N-Ag levels, found as a risk factor of severe Covid-19. Conclusions Two BNT162b2/Pfizer doses are associated with a 48% reduction of SARS-CoV-2 incidence and a 91.3% reduction of death risk in residents from NHs facing a VOC-α outbreak. BNT162b2/Pfizer efficacy was partly predicted by post-vaccine RBD-IgG levels.

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.2The 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. For t 2 is the most predominant,with 75% of midge-allergic patients showing specific IgE to For t 2.4 Allergic reactions to midge bites are not limited to humans but also seen in livestock, such as horses, cattle, sheep, and donkeys, causing significant veterinarian problems.E.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). The therapeutic approach is to imitate ASIT in human with established allergy while the prophylactic approach to non-allergics. Twenty-five μg For t 2 DNA was determined as the optimal dose after dose-finding experiments (Supplementary Fig S1). 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 hoursand had significantly increased at 48 hours after last treatment (Fig S3). Scratch bouts after rFor t 2 challenge were used as a clinical surrogate of itch. We measured For t 2-specific IgE, IgG1 and 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.After epicutaneous DNA vaccination, the allergen-induced itchin both groups significantly improved, and For t 2-specific IgE and IgG1/IgG2a ratio decreased significantly at week 6 or week 8 (Fig 2). Levels of mRNA and protein of IL-13 decreased significantly, but IFN-gamma and IL-10 remained unchanged. Expression of FOXP3 mRNA increased (Fig 2, protein data not shown). Eosinophils infiltration in the challenged skin significantly decreased (Fig S4).This is the first study to demonstrate an epicutaneous needless anti-allergic DNA vaccine that effectively treats an established allergic condition and prevents the development of an allergic disease using biting midge allergy as a model. After epicutaneous DNA vaccination, in addition to allergen-induced itch, the changes of multiple biomarkers suggest that immune tolerance was induced after 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 can penetrate the dermal barrier and translate the corresponding protein in the targeted skin and 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 potentially be used 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

Background: Although FoxP3 + regulatory T (Treg) cells constitute a highly heterogeneous population, with different regulatory potential depending on the disease context, distinct subsets or phenotypes remain poorly defined. This hampers the development of immunotherapy for allergic and autoimmune disorders. Objective: This study aimed at characterizing distinct FoxP3 + Treg subpopulations involved in the suppression of Th2-mediated allergic inflammation in the lung. Methods: We used an established mouse model of allergic airway disease based on ovalbumin sensitization and challenge to analyze FoxP3 + Tregs during the induction and resolution of inflammation, and identify markers that distinguish their most suppressive phenotypes. We also developed a new knock-in mouse model ( Foxp3creCd103dtr) enabling the specific ablation of CD103 +FoxP3 + Tregs for functional studies. Results: We found that during resolution of allergic airway inflammation in mice >50% of FoxP3 + Treg cells expressed the integrin CD103 which marks FoxP3 + Treg cells of high IL-10 production, increased expression of immunoregulatory molecules such as KLRG1, ICOS and CD127, and enhanced suppressive capacity for Th2-mediated inflammatory responses. CD103 +FoxP3 + Tregs were essential for keeping allergic inflammation under control as their specific depletion in Foxp3creCd103dtr mice lead to severe alveocapillary damage, eosinophilic pneumonia, and markedly reduced lifespan of the animals. Conversely, adoptive transfer of CD103 +FoxP3 + Tregs effectively treated disease, attenuating Th2 responses and allergic inflammation in an IL-10-dependent manner. Conclusion: Our study identifies a novel regulatory T cell population, defined by CD103 expression, programmed to prevent exuberant type 2 inflammation and keep homeostasis in the respiratory tract under control. This has important therapeutic implications.

Background: Serological tests are a powerful tool in the monitoring of infectious diseases and the detection of host immunity. However, manufacturers often provide diagnostic accuracy data generated through biased studies and the performance in clinical practice is essentially unclear.  Objectives: We aimed to determine the diagnostic accuracy of various serological testing strategies for (a) identification of patients with previous coronavirus disease-2019 (COVID-19) and (b) prediction of neutralizing antibodies against SARS-CoV-2 in real-life clinical settings.  Methods: We prospectively included 2’573 consecutive health-care workers and 1’085 inpatients with suspected or possible previous COVID-19 at a Swiss University Hospital. Various serological immunoassays based on different analytical techniques (enzyme-linked immunosorbent assays, ELISA; chemiluminescence immunoassay, CLIA; electrochemiluminescence immunoassay, ECLIA; lateral-flow immunoassay, LFI), epitopes of SARS-CoV-2 (nucleocapsid, N; receptor-binding domain, RBD; extended RBD, RBD+; S1 or S2 domain of the spike [S] protein, S1/S2), and antibody subtypes (IgG, pan-Ig) were conducted. A positive real-time PCR test from a nasopharyngeal swab was defined as previous COVID-19. Neutralization assays with live SARS-CoV-2 were performed in a subgroup of patients to assess neutralization activity (n=201).  Results: The sensitivity to detect patients with previous COVID-19 was ≥85% in anti-N ECLIA (86.8%) and anti-S1 ELISA (86.2%). Sensitivity was 84.7% in anti-S1/S2 CLIA, 84.0% in anti-RBD+ LFI, 81.0% in anti-N CLIA, 79.2% in anti-RBD ELISA, and 65.6% in anti-N ELISA. The specificity was 98.4% in anti-N ECLIA, 98.3% in anti-N CLIA, 98.2% in anti-S1 ELISA, 97.7% in anti-N ELISA, 97.6% in anti-S1/S2 CLIA, 97.2% in anti-RBD ELISA, and 96.1% in anti-RBD+ LFI. The sensitivity to detect neutralizing antibodies was ≥85% in anti-S1 ELISA (92.7%), anti-N ECLIA (91.7%), anti-S1/S2 CLIA (90.3%), anti-RBD+ LFI (87.9%), and anti-RBD ELISA (85.8%). Sensitivity was 84.1% in anti-N CLIA, and 66.2% in anti-N ELISA. The specificity was ≥97% in anti-N CLIA (100%), anti-S1/S2 CLIA (97.7%), and anti-RBD+ LFI (97.9%). Specificity was 95.9% in anti-RBD ELISA, 93.0% in anti-N ECLIA, 92% in anti-S1 ELISA, and 65.3% in anti-N ELISA. Diagnostic accuracy measures were consistent among subgroups.  Conclusions: The diagnostic accuracy of serological tests for SARS-CoV-2 antibodies varied remarkably in clinical practice, and the sensitivity to identify patients with previous COVID-19 deviated substantially from the manufacturer’s specifications. The data presented here should be considered when using such tests to estimate the infection burden within a specific population and determine the likelihood of protection against re-infection.

Allergen immunotherapy (AIT) has gained a permanent place in the therapeutic arsenal for the patient with allergy. Particularly, substantial evidence has been established for the efficacy of AIT in allergic rhinitis. A hallmark of AIT is it disease modifying effect resulting in persistent benefit after the treatment has been terminated. Both the subcutaneous and sublingual mode of administration appear to be safe. It is, however, a matter of debate whether AIT can be implemented for patients with asthma. EAACI and GINA guidelines recommend sublingual AIT in house dust mite driven asthma. The question however remains whether the different available forms of AIT should be used for allergic asthma in general.

COVID-19 Vaccines Tolerated in Patients with Paclitaxel and Docetaxel Allergy

Mass cytometry-based identification of a unique T-cell signature in childhood allergic asthma Short title: T-cell signature in paediatric AA

Background: The administration of L-glutamine (Gln) suppresses allergic airway inflammation via the rapid upregulation of MAPK phosphatase (MKP)-1, which functions as a negative regulator of inflammation by deactivating p38 and JNK mitogen-activated protein kinases (MAPKs). However, the role of endogenous Gln remains to be elucidated. Therefore, we investigated the mechanism by which endogenous Gln regulates MKP-1 induction and allergic airway inflammation in an ovalbumin-based murine asthma model. Methods: We depleted endogenous Gln levels using l-γ-glutamyl- p-nitroanilide (GPNA), an inhibitor of the Gln transporter ASCT2, and glutamine synthetase small interfering (si)RNA. Lentivirus expressing MKP-1 was injected to achieve overexpression of MKP-1. Asthmatic phenotypes were assessed using our previously developed ovalbumin-based murine model, which is suitable for examining sequential asthmatic events, including neutrophil infiltration. Gln levels were analyzed using a Gln assay kit. Results: GPNA or glutamine synthetase siRNA successfully depleted endogenous Gln levels. Importantly, homeostatic MKP-1 induction did not occur at all, which resulted in prolonged p38 MAPK and cytosolic phospholipase A 2 (cPLA 2) phosphorylation in Gln-deficient mice. Gln deficiency augmented all examined asthmatic reactions, but it exhibited a strong bias toward increasing the neutrophil count, which was not observed in MKP-1-overexpressing lungs. This neutrophilia was inhibited by a cPLA 2 inhibitor and a leukotriene B4 inhibitor, but not by dexamethasone. Conclusion: Gln deficiency leads to the impairment of MKP-1 induction and activation of p38 MAPK and cPLA 2, resulting in the augmentation of neutrophilic, more so than eosinophilic, airway inflammation.