not-yet-known not-yet-known not-yet-known unknown Background: IgE-mediated fish allergy has long been considered an umbrella term due to the high cross-reactivity of parvalbumin, the major fish allergen. Yet, clinical tolerance to certain fish highlights allergenicity differences. In this study, we sought to construct a fish allergenicity ladder and identify fish parvalbumin epitopes to improve the diagnosis of fish allergy. Methods: Reported clinical history and the serum-specific IgE (sIgE) responses of 200 fish allergic patients were collected and analyzed, while the relative parvalbumin content in different fish were measured for the construction of fish allergenicity ladder. Double-blind placebo-controlled food challenge (DBPCFC) and open challenge against salmon, grass carp and grouper were performed in 58 selected patients for validation of the ladder. Epitope mapping was performed by peptide array against parvalbumins of salmon (both β-1 and β-2), cod, grouper, and grass carp with sera from fish allergic (n=11), partial fish tolerant (n=12), and complete fish tolerant (n=5) patients diagnosed based on oral food challenge outcome. Results: The distribution pattern of clinical, sIgE and molecular data and their strong positive correlation led to the construction of a 4-step fish allergenicity ladder comprising: step 1 of the least allergenic fishes (tuna, halibut, salmon), steps 2 (cod) and 3 (herring and grouper) of moderately allergenic fishes to step 4 of highly allergenic fishes (catfish, grass carp and tilapia). Epitope mapping revealed one epitope from grouper parvalbumin (AA64-78) for diagnosing general fish allergy and one epitopic region from salmon parvalbumin (AA19-33) as biomarker of specific fish tolerance. Only epitope-specific IgE differentiated these patients but not sIgE to fish extract or parvalbumin. Conclusion: The fish ladder and epitopes discovery can precisely differentiate fish-allergic and tolerant subjects and guide fish reintroduction by stepping up the ladder, which innovate fish allergy care in the next millennium.

Editorial comment on the Special Issue “Omics in Food Allergy”Riccardo Castagnoli1,2, Ivan Taietti1,2,*, Agnes Sze-Yin Leung3, Philippe Eigenmann41. Pediatric Unit, Department of Clinical, Surgical, Diagnostic, and Pediatric Sciences, University of Pavia, Pavia, Italy2. Pediatric Clinic, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy3. Department of Paediatrics, Faculty of Medicine, The Chinese University of Hong Kong, China4. Pediatric Allergy Unit, Department of Pediatrics, Gynecology and Obstetrics, University Hospitals of Geneva, Geneva, SwitzerlandORCID:Riccardo Castagnoli: 0000-0003-0029-9383Ivan Taietti: 0000-0002-0372-523XAgnes Sze-Yin Leung: 0000-0001-8249-4478Philippe Eigenmann: 0000-0003-1738-1826Corresponding author:Ivan Taietti, MD;Pediatric Unit, Department of Clinical, Surgical, Diagnostic, and Pediatric Sciences, University of Pavia, 27100 Pavia, Italy.ivan.taietti@gmail.com; +39 340 7092400.Food allergy (FA), defined as an adverse reaction to food mediated by the immune system, is a significant public health issue with increasing prevalence over the past decades. Although FA affects up to 10% of children, the precise mechanisms underlying FA development and persistence remain inadequately understood1. Food immunotherapy has been successful in increasing the threshold of tolerance in individuals with persistent FA (desensitization), and in a subset, sustained unresponsiveness (remission) can be achieved. A combination of environmental and genetic factors is considered to be involved in the tolerance induction to food allergens. However, to date, it has been difficult to fully dissect the complexity of the biological determinants involved in FA.The emergence of “omics” sciences, including genomics, transcriptomics, proteomics, and metabolomics, is revolutionizing the basic and translational research approach, allowing for the comprehensive study of biological processes through analyzing and integrating multiple datasets from various biological perspectives.Moreover, epigenetic studies that evaluate the role of gene expression modifiers are deepening our understanding of the interaction between genetic predisposition and environmental influences.The Special Issue “Omics in Food Allergy” aims to present current knowledge and future perspectives in the field of FA through the lenses of omics sciences.Chun et al. and Lehmann et al. provide a detailed overview of epigenetic mechanisms involved in FA, including DNA methylation and microRNA (miRNAs)2,3. DNA methylation refers to the covalent addition of a methyl group, typically to a cytosine in a CpG dinucleotide in DNA. This limits access for transcription, frequently in the promoter region, leading to reduced gene expression. Moreover, miRNAs are small RNAs transcribed from intergenic or intronic genomic loci that decrease gene expression by mRNA degradation and translational inhibition. Of note, Chun et al. described two different approaches in investigating FA mechanisms: (i) the epigenome-wide research approach and (ii) candidate-gene investigations. The epigenome-wide research approach identified genomic regions differently methylated and regulated, involved in FA causation, antigen presentation, T cell development, and reaction severity. Candidate-gene investigations focused on Th1, Th2, T regulatory, and innate genes of interest in FA, highlighting the importance of methylation changes in specific candidate genes. In this context, for cow’s milk allergy (CMA), the attention is focused on novel genetic loci involved in Th1/Th2 differentiation pathways and potential B-cell dysfunction4. Of note, miRNA seems to be involved in CMA as demonstrated by the downregulation of mi193a-5p, a post-transcriptional regulator of IL-4 expression, in infants with CMA5. Moreover, methylation levels in peripheral blood mononuclear cells for IL-4, IL-5, IL-10, and IFN-γ negatively correlate with their respective serum cytokine concentrations and vary according to CMA status (active, resolved, or non-CMA). Finally, Chun et al. reviewed ongoing research aimed at finding DNA methylation predictors of food challenges. Initial work based on the concept of epigenetic markers as biomarkers of disease and treatment demonstrated promise for applying DNA methylation markers to potentially improve how we assess and manage patients with FA, but further validation in larger cohorts and work to practically translate such biomarkers to clinical practice is advocated2.Metabolomics may reflect the integration of genetic, transcriptomic, and proteomic variations with environmental factors, thus reflecting molecular processes of diseases. A metabolome approach would facilitate the identification of surrogate metabolite markers correlating with the disease activity and prognosis. Lee et al. reported an updated overview of metabolomics’s current application in the FA field. Alterations to the gut microbiota induced by Western dietary patterns are likely to profoundly affect host immunity and play a significant role in FA. Metabolites of interest include short-chain fatty acids (SCFAs), bile acid metabolites, and tryptophan metabolites due to their essential roles in normal immune development and homeostasis. SCFAs appear to be reduced in FA patients. Of note, it has been shown that bacterial SCFAs could protect against FA as a consequence of a high-fiber diet in mice. Accordingly, a reduction in Bifidobacterium during infancy, with a consequent reduction in SCFA levels is associated with an increased risk of allergy6. In peanut allergy, oral dysbiosis, reduced oral SCFA levels, and increased oral mucosal Th2 cytokine secretion characterize patients affected, and low fecal SCFAs at a young age could raise the risk of developing FA. Bile acid levels in stool seem to be involved in FA development and persistence, but blood level measurements are advocated to better characterize their role. Moreover, sphingolipids may modulate the function of invariant natural killer T cells and contribute to maintaining intestinal balance and protecting against FA through the modulation of the mast cells’ responsiveness. Sphingolipid dysmetabolism is involved in FA development as a consequence of immune dysregulation in modulating the differentiation of regulatory T cells and Th17 cells that is relevant in FA. Interestingly, some fecal sphingolipids seem to confer protection against FA while reduction of other of them seems to be associated with increased risk of FA, likewise the reduction in serum sphingolipids. Unfortunately, no definitive evidence is available to distinguish whether the sphingolipids modulating this FA effect are dietary or microbial-derived. Metabolites of tryptophan from kynurenine and indole pathways are linked to the function of the intestinal barrier and mucosal immune responses (mainly T regulatory cells) and contribute to intestinal homeostasis. A reduction in these pathways is linked to multiple FA in children. To date, the role of omega-3-polyunsaturated fatty acids metabolites is not clearly defined in FA. However, 17-hydroxy-docosahexaenoic acid (17-HDHA) has been reported to inhibit IgE production by B cells and to suppress the differentiation of naïve B cells into IgE-secreting cells7.The gut microbiome (GM) and its metabolic product, as previously mentioned, play an important role in FA development. TheBifidobacteriaceae family and bacteria of theLactobacillales order are consistently reported lower in CMA children. At the same time, the Firmicutes phylum, primarily associated with the Clostridia class, is consistently increased in mice and human studies. It seems discordant with the observation that infants with resolved CMA were reported to have enrichedClostridia class at 3-6 months but with non-conclusive data. However, Savova et al. showed that GM with enriched Clostridiaclass, reduced Lactobacillales order, and reducedBifidobacterium genus is associated with CMA in early life. Emerging evidence shows that prebiotics, probiotics, and symbiotics, may be a promising adjuvant in promoting tolerance acquisition. ElevatedBifidobacterium genus and reduced Clostridia class members were consistently observed post-treatment with Bifidobacteriumstrains probiotics or after lactose-supplemented extensively hydrolyzed formula (EHF) treatment strategy but with decreased levels ofLactobacillus genus in those treated with Bifidobacteriumstrains probiotics. Gaps remain in understanding the relationship between microbiome and immune response and between transcriptomics (including genes related to the immune response) and GM. In vivo ,Bifidobacterium bifidum has been shown to reduce allergy symptoms, lower serum IgE and raise IgG2 levels, decrease the pro-inflammatory cytokines (TNFα, IL- 1β, and IL-6), and increase the anti-inflammatory cytokine IL-10 in CMA-patients8. Recently, fiber diet has been shown to modulate the disease course of FA through diet-driven changes in the GM9. It is necessary to conduct further research on the impact of GM on FA development and persistence.Lastly, oral immunotherapy (OIT) may provide an active treatment that enables to increase the amount of food that the patient can intake without reaction during treatment (i.e., desensitization), and reduces the risk of potential life-threatening allergic reaction in the event of accidental ingestion10 Unfortunately, there are still significant gaps in understanding the immune mechanisms following OIT, but increasing knowledge about transcriptional pathways associated with its outcomes is available to date11. Ashley et al.12 show that OIT-induced remission of FA is linked to the anergic T cell state mediated by anergy of memory T cells associated with mantained T regulatory cell activity. Suppression of the Th2 transcriptional signals in Th2A-like cells was linked to desensitization following OIT. Moreover, the dampened Th2 and Th1 signatures in effector cells are linked with FA remission, while baseline inflammatory signals in Th1 and Th17 effector cells were associated with poorer outcomes following OIT. Type I interferons were recently identified as potential regulators of remission following OIT because of their key role in the suppression of the Th2 antigen response through regulatory action on GATA3 and the high-affinity IgE receptor. Moreover, an early transient increase in TGF-β producing cells one year into treatment seems to be associated with good clinical outcomes. In addition, persistent activation of FOXP3, expressed by T regulatory cells, may be a critical requirement for lasting persistence of remission12.Comprehensive multi-omics studies are essential to understand FA mechanistically, as highlighted by this Special Issue. “Omics” studies of FA are of great interest as they allow for a thorough understanding of the complexity of FA development and therapeutic outcomes. This has a fundamental impact on approaches to precision medicine.

Food allergy is postulated to originate from cutaneous sensitization through a disrupted skin barrier, particularly in atopic dermatitis (AD). Strategies for food allergy prevention currently centre around early allergic food introduction, but there is now increasing evidence for the role of early skin barrier restoration in the form of prophylactic emollient therapy and early aggressive, proactive treatment of established AD for food allergy prevention. Research gaps that remain to be addressed include the type of emollient or anti-inflammatory medication which confers the greatest efficacy in preventive or proactive skin treatment respectively, the duration of therapy, and the window of opportunity for these interventions.

Background: Recent studies indicated that fish-allergic patients may safely consume certain fish species. Multiplex IgE testing facilitates the identification of species tolerated by individual patients. Methods: Sera were collected from 263 fish-allergic patients from Austria, China, Denmark, Luxembourg, Norway and Spain. Specific (s) IgE to parvalbumins (PVs) from 10 fish species along with IgE to 7 raw and 6 heated fish extracts was quantified using a research version of the ALEX 2 assay. IgE-signatures of individual patients and patient groups were analyzed using SPSS and R. Results: sIgE to alpha-PV from ray, a cartilaginous fish, was not detected in 78% of the patients while up to 41% of the patients, depending on their country of origin, tested negative for at least one beta-PV. sIgE values were highest for mackerel and tuna PVs (>10 kUA/L) and significantly lower for cod (4.9 kUA/L) and sole PVs (2.55 kUA/L). 17% of the patients, although negative for PVs, tested positive for the respective fish extracts. Based on the absence of IgE to PVs and extracts, up to 21% of the patients were identified as potentially tolerating one or more bony fish. Up to 90% of the patients tested negative for ray. The probability of negativity to one fish based on negativity to others was calculated. Negativity to tuna and mackerel emerged as a good marker of negativity to additional bony fish. Conclusion: Measuring sIgE to PVs and extracts from evolutionary distant fish species indicates bony and cartilaginous fish species for tolerance-confirming food challenges.

Background The current diagnostics of fish allergy lack sufficient accuracy such that more reliable tests such as component-resolved diagnosis (CRD) are urgently needed. This study aimed at identifying fish allergens of salmon and grass carp and evaluating the sensitization pattern towards the identified allergens in fish allergic subjects from two distinct populations in Asia. Methods One hundred and three fish allergic subjects were recruited from Hong Kong (67 subjects) and Japan (46 subjects). Western blot and mass spectrometry were used to identify allergens from salmon and grass carp. Fish allergens were purified and tested against 96 sera on ELISA to analyze patients’ sensitization pattern. The protein profiles of salmon meat prepared under different cooking methods until core temperature reached 80°C were evaluated by SDS-PAGE and mass spectrometry. Results Three common allergens between salmon and grass carp, namely enolase, glycerldehyde-3-phosphate dehydrogenase (GAPDH) and parvalbumin, and two salmon-specific allergens collagen and aldolase were identified. Parvalbumin was the major allergen for both fishes showing an overall sensitization rate of 74.7%, followed by collagen (38.9%), aldolase (38.5%) and enolase (17.8%). Japanese subjects showed more diverse allergen sensitization pattern and more frequent IgE-binding to heat-labile salmon allergens. Compared with steaming and boiling, cooking by baking and frying retained more fish proteins inclusive of heat-labile allergens. Conclusions Fish allergic patients from different Asian populations show varying fish allergen sensitization profiles. The relevant extracts and components for diagnosis are population-dependent but parvalbumin and collagen are important biomarkers. Cooking methods modify allergen composition of salmon and appear to influence patients’ allergic manifestations.

Background: Anaphylaxis is a significant health burden in most Western countries but there is little published data on the incidence and pattern of anaphylaxis in Asia. We aim to determine the incidence rate and pattern of anaphylaxis over the past decade among the paediatric population in Hong Kong. Methods: Medical records of patients presenting with allergy-related symptoms during the period 2010 to 2019 were examined. Paediatric patients aged below 18 years who fulfilled the diagnostic criteria for anaphylaxis laid out by the NIAID/FAAN were analysed. Incidence rates were calculated using population statistics as the denominator. All information pertaining to the anaphylaxis events and patients’ characteristics were retrieved using standardized data collection forms. Results: The overall 10-year estimated incidence of anaphylaxis was 7.70 per 100,000 person-years, with a rising trend of anaphylaxis incidence across time. Food-induced anaphylaxis accounted for the majority of hospital presentation, of which peanut and shellfish were the top food triggers in our population. Comorbid asthma and young age were risk factors associated with wheeze at presentation. Misdiagnosis of anaphylaxis occurred in up to half the anaphylaxis cases and adrenaline was only utilised in 45% of cases. Conclusions: An increasing trend of anaphylaxis incidence over the past decade is evident in Hong Kong children, with a discrepantly low accuracy in diagnosis and suboptimal management of anaphylaxis. There is a pressing need to heighten public and physicians’ awareness of the distinctive features of anaphylaxis in the paediatric age group.

Background: Combined treatment with probiotic and peanut oral immunotherapy (PPOIT) was shown to induce sustained unresponsiveness (SU) in a proof-of-concept randomized trial. Additional data on safety and long-term outcomes are needed. This study aimed to evaluate the safety and long-term effects of PPOIT in children with peanut allergy. Methods: Open-label study of 20 children aged 1-12 years with challenge-confirmed peanut allergy; all children received 18-months of PPOIT. Efficacy endpoints were desensitization, 8-week SU, and persistence of 8-week SU at 3-years post-treatment, assessed by double-blind placebo-controlled food challenge (cumulative 4950mg peanut protein). Treatment emergent adverse events and relationship to study treatment were recorded. Immunologic measures and health related quality of life (HRQL) were evaluated at screening, end-of-treatment and 3-years post-treatment. Results: Sixteen children (75%) completed treatment. By intention-to-treat analysis, 75% (15/20) achieved desensitization and 60% (12/20) achieved 8-week SU. Ten of 12 participants with SU at end-of-treatment consented to the 3-year SU challenge; 6 (60%) had persistence of SU. PPOIT was associated with significantly reduced peanut skin prick test wheal size and serum peanut specific-IgE levels at end-of-treatment, 12-months and 3-years post-treatment. There were no serious adverse events. HRQL scores improved (exceeding the Minimal Clinically Important Difference of 0.45) at 12-months post-treatment with benefit sustained at 3-years post-treatment. Conclusions: Eighteen months of PPOIT induced high rates of desensitization and SU, and SU persisted to 3-years post-treatment in a majority of initial responders. PPOIT led to long-lasting suppression of peanut sIgE and long-lasting clinically important improvement in HRQL.

Background: Anaphylaxis has been increasing in developed countries but there is very little published data on the burden of anaphylaxis and the pattern of adrenaline autoinjector (AAI) prescription from Asia. Objectives: We aim to determine the incidence rates of anaphylaxis and prescription rates of AAI over the past decade in Hong Kong. Methods: Using a centralized electronic database of Hong Kong’s sole public-funded healthcare provider, we obtained and analysed all patients between 2009 and 2019 with physician-reported diagnosis of anaphylaxis. Incidence rates were calculated using population statistics as the denominator. Patients’ prescriptions on discharge were collected to determine the AAI prescription rates. Results: The overall 10-year estimated incidence rate of anaphylaxis was 3.57 per 100,000 person-years. An increasing trend over time across both pediatric and adult populations from 2009 to 2019 was found which was more marked among the pediatric population. There was an overall increasing rates of AAI prescription for patients admitted for anaphylaxis, but the overall AAI prescription rate was less than 15% and was significantly less likely to be prescribed for the adult compared to pediatric patients (36.5% vs. 89.4%, p<0.001). Conclusions: An increasing trend of anaphylaxis incidence rates over the past decade is evident in Asian populations, with a discrepantly low rate of AAI prescription, particularly in the adult patients.