Saliva antibody profiles are associated with reaction threshold and severity of peanut allergic reactions.

BACKGROUND: Reaction threshold and severity in food allergy are difficult to predict, and noninvasive predictors are lacking. OBJECTIVE: We sought to determine the relationships between pre-challenge levels of peanut (PN)-specific antibodies in saliva and reaction threshold, severity, and organ-specific symptoms during PN allergic reactions. METHODS: We measured PN-specific antibody levels in saliva collected from 127 children with suspected PN allergy before double-blind, placebo-controlled PN challenges in which reaction threshold, severity, and symptoms were rigorously characterized. Low threshold (LT) PN allergy was defined as reaction to <300 mg of PN protein cumulatively consumed. A consensus severity grading system was used to grade severity. We analyzed associations between antibody levels and reaction threshold, severity, and organ-specific symptoms. RESULTS: Among the 127 children, those with high pre-challenge saliva PN IgE had higher odds of LT PN allergy (odds ratio [OR] 3.9, 95% CI 1.6-9.5), while those with high saliva PN IgA:PN IgE ratio or PN IgG4:PN IgE ratio had lower odds of LT PN allergy (OR 0.3, 95% CI 0.1-0.8; OR 0.4, 95% CI 0.2-0.9). Children with high pre-challenge saliva PN IgG4 had lower odds of severe PN reactions (OR 0.4, 95% CI 0.2-0.9). Children with high saliva PN IgE had higher odds of respiratory symptoms (OR 8.0, 95% CI 2.2-26.8). Saliva PN IgE modestly correlated with serum PN IgE levels (Pearson r = 0.31, P = .0004). High and low saliva PN IgE levels further distinguished reaction threshold and severity in participants stratified by serum PN IgE, suggesting endotypes. CONCLUSIONS: Saliva PN antibodies could aid in noninvasive risk stratification of PN allergy threshold, severity, and organ-specific symptoms.

Utility of epitope-specific IgE, IgG4, and IgG1 antibodies for the diagnosis of wheat allergy.

BACKGROUND: The bead-based epitope assay has been used to identify epitope-specific (es) antibodies and successfully used to diagnose clinical allergy to milk, egg, and peanut. OBJECTIVE: We sought to identify es-IgE, es-IgG4, and es-IgG1 of wheat proteins and determine the optimal peptides to differentiate wheat-allergic from wheat-tolerant using the bead-based epitope assay. METHODS: Children and adolescents who underwent an oral food challenge to confirm their wheat allergy status were enrolled. Seventy-nine peptides from α-/ß-gliadin, γ-gliadin, ω-5-gliadin, and high- and low-molecular-weight glutenin were commercially synthesized and coupled to LumAvidin beads (Luminex Corporation, Austin, Tex). Machine learning methods were used to identify diagnostic epitopes, and performance was evaluated using the DeLong test. RESULTS: The analysis included 122 children (83 wheat-allergic and 39 wheat-tolerant; 57.4% male). Machine learning coupled with simulations identified wheat es-IgE, but not es-IgG4 or es-IgG1, to be the most informative for diagnosing wheat allergy. Higher es-IgE binding intensity correlated with the severity of allergy phenotypes, with wheat anaphylaxis exhibiting the highest es-IgE binding intensity. In contrast, wheat-dependent exercise-induced anaphylaxis showed lower es-IgG1 binding intensity than did all the other groups. A set of 4 informative epitopes from ω-5-gliadin and γ-gliadin were the best predictors of wheat allergy, with an area under the curve of 0.908 (sensitivity, 83.4%; specificity, 88.4%), higher than the performance exhibited by wheat-specific IgE (area under the curve = 0.646; P < .001). The predictive ability of our model was confirmed in an external cohort of 71 patients (29 allergic, 42 nonallergic), with an area under the curve of 0.908 (sensitivity, 75.9%; specificity, 90.5%). CONCLUSIONS: The wheat bead-based epitope assay demonstrated greater diagnostic accuracy compared with existing specific IgE tests for wheat allergy.

Joint transcriptomic and cytometric study of children with peanut allergy reveals molecular and cellular cross talk in reaction thresholds.

BACKGROUND: Reaction thresholds in peanut allergy are highly variable. Elucidating causal relationships between molecular and cellular processes associated with variable thresholds could point to therapeutic pathways for raising thresholds. OBJECTIVE: The aim of this study was to characterize molecular and cellular systemic processes associated with reaction threshold in peanut allergy and causal relationships between them. METHODS: A total of 105 children aged 4 to 14 years with suspected peanut allergy underwent double-blind, placebo-controlled food challenge to peanut. The cumulative peanut protein quantity eliciting allergic symptoms was considered the reaction threshold for each child. Peripheral blood samples collected at 0, 2, and 4 hours after challenge start were used for RNA sequencing, whole blood staining, and cytometry. Statistical and network analyses were performed to identify associations and causal mediation between the molecular and cellular profiles and peanut reaction threshold. RESULTS: Within the cohort (N = 105), 81 children (77%) experienced allergic reactions after ingesting varying quantities of peanut, ranging from 43 to 9043 mg of cumulative peanut protein. Peripheral blood expression of transcripts (eg, IGF1R [false discovery rate (FDR) = 5.4e-5] and PADI4 [FDR = 5.4e-5]) and neutrophil abundance (FDR = 9.5e-4) were associated with peanut threshold. Coexpression network analyses revealed that the threshold-associated transcripts were enriched in modules for FcγR-mediated phagocytosis (FDR = 3.2e-3) and Toll-like receptor (FDR = 1.4e-3) signaling. Bayesian network, key driver, and causal mediation analyses identified key drivers (AP5B1, KLHL21, VASP, TPD52L2, and IGF2R) within these modules that are involved in bidirectional causal mediation relationships with neutrophil abundance. CONCLUSION: Key driver transcripts in FcγR-mediated phagocytosis and Toll-like receptor signaling interact bidirectionally with neutrophils in peripheral blood and are associated with reaction threshold in peanut allergy.

Multi-omic integration reveals alterations in nasal mucosal biology that mediate air pollutant effects on allergic rhinitis.

BACKGROUND: Allergic rhinitis is a common inflammatory condition of the nasal mucosa that imposes a considerable health burden. Air pollution has been observed to increase the risk of developing allergic rhinitis. We addressed the hypotheses that early life exposure to air toxics is associated with developing allergic rhinitis, and that these effects are mediated by DNA methylation and gene expression in the nasal mucosa. METHODS: In a case-control cohort of 505 participants, we geocoded participants' early life exposure to air toxics using data from the US Environmental Protection Agency, assessed physician diagnosis of allergic rhinitis by questionnaire, and collected nasal brushings for whole-genome DNA methylation and transcriptome profiling. We then performed a series of analyses including differential expression, Mendelian randomization, and causal mediation analyses to characterize relationships between early life air toxics, nasal DNA methylation, nasal gene expression, and allergic rhinitis. RESULTS: Among the 505 participants, 275 had allergic rhinitis. The mean age of the participants was 16.4 years (standard deviation = 9.5 years). Early life exposure to air toxics such as acrylic acid, phosphine, antimony compounds, and benzyl chloride was associated with developing allergic rhinitis. These air toxics exerted their effects by altering the nasal DNA methylation and nasal gene expression levels of genes involved in respiratory ciliary function, mast cell activation, pro-inflammatory TGF-ß1 signaling, and the regulation of myeloid immune cell function. CONCLUSIONS: Our results expand the range of air pollutants implicated in allergic rhinitis and shed light on their underlying biological mechanisms in nasal mucosa.

Citrin: a novel food allergen in citrus seeds and citrus-derived pectin that shows cross-reactivity with cashew and pistachio.

BACKGROUND: Patients exquisitely sensitive to cashew/pistachio are at risk for allergic reactions to citrus seeds and pectin. OBJECTIVE: In this study, we sought to evaluate whether pectin is contaminated with citrus seeds, to identify a culprit antigen in citrus seeds, and to assess for cross-reactivity among allergens in citrus seeds, citrus pectin, and cashew or pistachio. METHODS: Proteins from orange seed coats, orange seed endosperms, lemon seeds, grapefruit seeds, citrus pectin, apple pectin, and grapefruit pectin were extracted. Protein concentrations in all extracts were determined and visualized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique. Immunoglobulin E-binding capacity was determined with Western blot analyses and tandem mass spectrometry for the identification of the culprit allergen in citrus seeds and pectin. RESULTS: In subjects with citrus seed, pectin, and cashew allergies, there was strong immunoglobulin E-reactivity to bands between 17 to 28 kDa and 28 to 38 kDa. The tandem mass spectrometry analysis of these bands indicated the presence of citrin as the culprit allergen. Citrin and Ana o 2 are both 11S globulins belonging to the cupin superfamily, and significant homology was found between these proteins. CONCLUSION: Citrus pectin may be contaminated with citrus seeds. Citrin, a newly identified allergen in citrus seeds, seems to be the culprit antigen in citrus seeds and contaminated citrus pectin. Citrin is highly homologous with Ana o 2 in cashew and Pis v 2 in pistachio, suggesting potential for cross-reactivity and providing an explanation for co-allergenicity of cashew or pistachio, citrus seeds, and citrus pectin.

Examination of host genetic effects on nasal microbiome composition.

BACKGROUND: Genetic predisposition increases risk for asthma, and distinct nasal microbial compositions are associated with asthma. Host genetics might shape nasal microbiome composition. OBJECTIVE: We examined associations between host genetics and nasal microbiome composition. METHODS: Nasal samples were collected from 584 participants from the Mount Sinai Health System, New York. Seventy-seven follow-up samples were collected from a subset of 40 participants. 16S rRNA sequencing and RNA sequencing were performed on nasal samples. Beta diversity was calculated, variant calling on RNA sequencing data was performed, and genetic relatedness between individuals was determined. Using linear regression models, we tested for associations between genetic relatedness and nasal microbiome composition. RESULTS: The median age of the cohort was 14.6 (interquartile range 11.2-19.5) years, with participants representing diverse ancestries and 52.7% of the cohort being female. For participants who provided follow-up samples, the median time between samples was 5.1 (interquartile range 1.4-7.2) months. Nasal microbiome composition similarity as reflected by beta diversity was significantly higher within subjects over time versus between subjects (coefficient = 0.091, P = 2.84-7). There was no significant association between genetic relatedness and beta diversity (coefficient = -0.05, P = .29). Additional analyses exploring the relationship between beta diversity and genetic variance yielded similar results. CONCLUSION: Host genetics has little influence on nasal microbiome composition.

Mapping Sequential IgE-Binding Epitopes on Major and Minor Egg Allergens.

INTRODUCTION: Molecular studies of hen's egg allergens help define allergic phenotypes, with IgE to sequential (linear) epitopes on the ovomucoid (OVM) protein associated with a persistent disease. Epitope profiles of other egg allergens are largely unknown. The objective of this study was to construct an epitope library spanning across 7 allergens and further evaluate sequential epitope-specific (ses-)IgE and ses-IgG4 among baked-egg reactive or tolerant children. METHODS: A Bead-Based Epitope Assay was used to identify informative IgE epitopes from 15-mer overlapping peptides covering the entire OVM and ovalbumin (OVA) proteins in 38 egg allergic children. An amalgamation of 12 B-cell epitope prediction tools was developed using experimentally identified epitopes. This ensemble was used to predict epitopes from ovotransferrin, lysozyme, serum albumin, vitellogenin-II fragment, and vitellogenin-1 precursor. Ses-IgE and ses-IgG4 repertoires of 135 egg allergic children (82 reactive to baked-egg, the remaining 52 tolerant), 46 atopic controls, and 11 healthy subjects were compared. RESULTS: 183 peptides from OVM and OVA were screened and used to create an aggregate algorithm, improving predictions of 12 individual tools. A final library of 65 sequential epitopes from 7 proteins was constructed. Egg allergic children had higher ses-IgE and lower ses-IgG4 to predominantly OVM epitopes than both atopic and healthy controls. Baked-egg reactive children had similar ses-IgG4 but greater ses-IgE than tolerant group. A combination of OVA-sIgE with ses-IgEs to OVM-023 and OVA-028 was the best predictor of reactive phenotype. CONCLUSION: We have created a comprehensive epitope library and showed that ses-IgE is a potential biomarker of baked-egg reactivity.

The nasal microbiome, nasal transcriptome, and pet sensitization.

BACKGROUND: Pet allergies are common in children with asthma. Microbiota and host responses may mediate allergen sensitization. OBJECTIVE: We sought to uncover host-microbe relationships in pet allergen sensitization via joint examination of the nasal microbiome and nasal transcriptome. METHODS: We collected nasal samples from 132 children with asthma for parallel 16S rRNA and RNA sequencing. Specific IgE levels for cat and dog dander were measured. Analyses of the nasal microbiome, nasal transcriptome, and their correlations were performed with respect to pet sensitization status. RESULTS: Among the 132 children, 91 (68.9%) were cat sensitized and 96 (72.7%) were dog sensitized. Cat sensitization was associated with lower nasal microbial diversity by Shannon index (P = .021) and differential nasal bacterial composition by weighted UniFrac distance (permutational multivariate ANOVA P = .035). Corynebacterium sp and Staphylococcus epidermidis were significantly less abundant, and the metabolic process "fatty acid elongation in mitochondria" was lower in pet-sensitized versus unsensitized children. Correlation networks revealed that the nasal expression levels of 47 genes representing inflammatory processes were negatively correlated with the relative abundances of Corynebacterium sp and S epidermidis. Thus, these species were directly associated not only with the absence of pet sensitization but also with the underexpression of host gene expression of inflammatory processes that contribute to allergen sensitization. Causal mediation analyses revealed that the associations between these nasal species and pet sensitization were mediated by nasal gene expression. CONCLUSIONS: Higher abundances of nasal Corynebacterium sp and S epidermidis are associated with absence of pet sensitization and correlate with lower expression of inflammatory genes.

Network study of nasal transcriptome profiles reveals master regulator genes of asthma.

BACKGROUND: Nasal transcriptomics can provide an accessible window into asthma pathobiology. OBJECTIVE: Our goal was to move beyond gene signatures of asthma to identify master regulator genes that causally regulate genes associated with asthma phenotypes. METHODS: We recruited 156 children with severe persistent asthma and controls for nasal transcriptome profiling and applied network-based and probabilistic causal methods to identify severe asthma genes and their master regulators. We then took the same approach in an independent cohort of 190 adults with mild/moderate asthma and controls to identify mild/moderate asthma genes and their master regulators. Comparative analysis of the master regulator genes followed by validation testing in independent children with severe asthma (n = 21) and mild/moderate asthma (n = 154) was then performed. RESULTS: Nasal gene signatures for severe persistent asthma and for mild/moderate persistent asthma were identified; both were found to be enriched in coexpression network modules for ciliary function and inflammatory response. By applying probabilistic causal methods to these gene signatures and validation testing in independent cohorts, we identified (1) a master regulator gene common to asthma across severity and ages (FOXJ1); (2) master regulator genes of severe persistent asthma in children (LRRC23, TMEM231, CAPS, PTPRC, and FYB); and (3) master regulator genes of mild/moderate persistent asthma in children and adults (C1orf38 and FMNL1). The identified master regulators were statistically inferred to causally regulate the expression of downstream genes that modulate ciliary function and inflammatory response to influence asthma. CONCLUSION: The identified master regulator genes of asthma provide a novel path forward to further uncovering asthma mechanisms and therapy.

Accurate and reproducible diagnosis of peanut allergy using epitope mapping.

BACKGROUND: Accurate diagnosis of peanut allergy is a significant clinical challenge. Here, a novel diagnostic blood test using the peanut bead-based epitope assay ("peanut BBEA") was developed utilizing the LEAP cohort and then validated using two independent cohorts. METHODS: The development of the peanut BBEA diagnostic test followed the National Academy of Medicine's established guidelines with discovery performed on 133 subjects from the non-interventional arm of the LEAP trial and an independent validation performed on 82 subjects from the CoFAR2 and 84 subjects from the POISED study. All samples were analyzed using the peanut BBEA methodology, which measures levels of IgE to two Ara h 2 sequential (linear) epitopes and compares their combination to a threshold pre-specified in the model development phase. When a patient has an inconclusive outcome by skin prick testing (or sIgE), IgE antibody levels to this combination of two epitopes can distinguish whether the patient is "Allergic" or "Not Allergic." Diagnoses of peanut allergy in all subjects were confirmed by double-blind placebo-controlled food challenge and subjects' ages were 7-55 years. RESULTS: In the validation using CoFAR2 and POISED cohorts, the peanut BBEA diagnostic test correctly diagnosed 93% of the subjects, with a sensitivity of 92%, specificity of 94%, a positive predictive value of 91%, and negative predictive value of 95%. CONCLUSIONS: In validation of the peanut BBEA diagnostic test, the overall accuracy was found to be superior to existing diagnostic tests for peanut allergy including skin prick testing, peanut sIgE, and peanut component sIgE testing.

Endotoxin, food allergen sensitization, and food allergy: A complementary epidemiologic and experimental study.

BACKGROUND: Household endotoxin levels have been variably associated with risk for asthma and atopy. METHODS: We studied participants from the 2005-2006 National Health and Nutrition Examination Survey (NHANES, n = 6963), a large cohort representative of the US population (aged 1-84 years). We built logistic regression models to test for associations between house dust endotoxin and sensitization to specific foods (milk, egg, and peanut). To experimentally explore the detected epidemiologic associations, peripheral blood mononuclear cells (PBMCs) were collected from 21 children (aged 1-19 years) mono-food allergic (ie, sensitized and clinically reactive) to milk, egg, or peanut and nonallergic controls for stimulation with endotoxin and secreted cytokine measurement. For each food allergy, linear mixed-effects models were built to test the association between endotoxin stimulation and cytokine level. RESULTS: Among NHANES subjects, the geometric mean household endotoxin level was 15.5 EU/mg (GSE 0.5). Prevalence of food allergen sensitization (sIgE ≥ 0.35 kUA /L) varied by food: milk 5.7%, egg 4.0%, and peanut 7.9%. In models adjusted for potential confounders (age, race, country of birth, total people per household, US region, and history of wheezing in the past year), household endotoxin level was associated with sensitization to milk (OR 1.7, 95% CI 1.2-2.1) and egg (OR 1.4, 95% CI 1.01-1.9), but not peanut (OR 0.98, 95% CI 0.8-1.2). Interferon-γ levels of endotoxin-stimulated PBMCs from children allergic to milk or egg, but not peanut, were significantly lower compared to controls in linear mixed-effects models adjusted for repeated measures, experimental variables, age, and inter-individual variability (P-values .007, .018, and .058, respectively). CONCLUSION: Higher household endotoxin is associated with increased odds of milk and egg sensitization. Altered cytokine responsiveness to endotoxin is also observed in PBMCs from individuals with milk and egg allergy.

Ovomucoid epitope-specific repertoire of IgE, IgG4 , IgG1 , IgA1 , and IgD antibodies in egg-allergic children.

BACKGROUND: Egg-white ovomucoid, that is, Gal d 1, is associated with IgE-mediated allergic reactions in most egg-allergic children. Epitope-specific IgE levels have been correlated with the severity of egg allergy, while emerging evidence suggests that other antibody isotypes (IgG1 , IgG4 , IgA, and IgD) may have a protective function; yet, their epitope-specific repertoires and associations with atopic comorbidities have not been studied. METHODS: Bead-based epitope assay (BBEA) was used to quantitate the levels of epitope-specific (es)IgA, esIgE, esIgD, esIgG1 , and esIgG4 antibodies directed at 58 (15-mer) overlapping peptides, covering the entire sequence of ovomucoid, in plasma of 38 egg-allergic and 6 atopic children. Intraclass correlation (ICC) and coefficient of variation (CV) were used for the reliability assessment. The relationships across esIgs were evaluated using network analysis; linear and logistic regressions were used to compare groups based on egg allergy status and comorbidities. RESULTS: BBEA had high reliability (ICC >0.75) and low variability (CV <20%) and could detect known IgE-binding epitopes. Egg-allergic children had lower esIgA1 (P = .010) and esIgG1 (P = .016) and higher esIgE (P < .001) and esIgD (P = .015) levels compared to the atopic controls. Interestingly, within the allergic group, children with higher esIgD had decreased odds of anaphylactic reactions (OR =0.48, P = .038). Network analysis identified most associations between esIgE with either esIgG4 or esIgD; indicating that IgE-secreting plasma cells could originate from either sequential isotype switch from antigen-experienced intermediate isotypes or directly from the IgD+ B cells. CONCLUSIONS: Collectively, these data point toward a contribution of epitope-specific antibody repertoires to the pathogenesis of egg allergy.

IgE and IgG4 binding to lentil epitopes in children with red and green lentil allergy.

BACKGROUND: The consumption of lentil is common in the Mediterranean area and is one of the causes of IgE-mediated food allergy in many countries. Len c 1 is a well-defined allergen of lentil and approximately 80% of the patients with lentil allergy recognize the purified Len c 1 protein. We sought to identify IgE and IgG4 sequential epitopes of Len c 1 in patients with red and/or green lentil allergy. We also aimed to determine IgE and IgG4 binding differences between those patients who had outgrown or remained reactive to lentil. METHODS: Children with IgE-mediated lentil allergy were included in the study. We applied a microarray immunoassay to determine the characterization of positive IgE and IgG4 binding to Len c 1 epitopes in the patients' sera. RESULTS: The peptides specifically recognized by IgE and IgG4 antibodies were mainly detected between peptides 107 and 135 of Len c 1. The signal intensities of positive epitopes were significantly greater in reactive patients than tolerant ones (P = .008 for IgE and P = .002 for IgG4). Moreover, IgE and IgG4 antibodies bound largely the same sequential epitopes in patients who remained reactive or outgrew their allergy. CONCLUSION: IgG4-binding epitopes in lentil allergy were identified and IgE and IgG4 binding to epitopes in both red and green lentils was compared. Our data regarding signal intensity differences between reactive and outgrown patients and overlap binding of IgE and IgG4 antibodies may be important for the development of more accurate diagnostic tests and understanding of natural tolerance development.

Predicting development of sustained unresponsiveness to milk oral immunotherapy using epitope-specific antibody binding profiles.

BACKGROUND: In a recent trial of milk oral immunotherapy (MOIT) with or without omalizumab in 55 patients with milk allergy treated for 28 months, 44 of 55 subjects passed a 10-g desensitization milk protein challenge; 23 of 55 subjects passed the 10-g sustained unresponsiveness (SU) challenge 8 weeks after discontinuing MOIT. OBJECTIVE: We sought to determine whether IgE and IgG4 antibody binding to allergenic milk protein epitopes changes with MOIT and whether this could predict the development of SU. METHODS: By using a novel high-throughput Luminex-based assay to quantitate IgE and IgG4 antibody binding to 66 sequential epitopes on 5 milk proteins, serum samples from 47 subjects were evaluated before and after MOIT. Machine learning strategies were used to predict whether a subject would have SU after 8 weeks of MOIT discontinuation. RESULTS: MOIT profoundly altered IgE and IgG4 binding to epitopes, regardless of treatment outcome. At the initiation of MOIT, subjects achieving SU exhibited significantly less antibody binding to 40 allergenic epitopes than subjects who were desensitized only (false discovery rate ≤ 0.05 and fold change > 1.5). Based on baseline epitope-specific antibody binding, we developed predictive models of SU. Using simulations, we show that, on average, IgE-binding epitopes alone perform significantly better than models using standard serum component proteins (average area under the curve, >97% vs 80%). The optimum model using 6 IgE-binding epitopes achieved a 95% area under the curve and 87% accuracy. CONCLUSION: Despite the relatively small sample size, we have shown that by measuring the epitope repertoire, we can build reliable models to predict the probability of SU after MOIT. Baseline epitope profiles appear more predictive of MOIT response than those based on serum component proteins.

A new Luminex-based peptide assay to identify reactivity to baked, fermented, and whole milk.

BACKGROUND: The majority of children with cow's milk allergy (CMA) tolerate baked milk. However, reactivity to fermented milk products such as yogurt/cheese has not been previously evaluated. We sought to determine whether children with CMA could tolerate yogurt/cheese and whether a patient's IgE and IgG4-binding pattern to milk protein epitopes could distinguish clinical reactivity. METHODS: Four groups of reactivity were identified by Oral food challenge: baked milk reactive, fermented milk reactive, whole milk reactive, and outgrown. sIgE and sIgG4 binding to milk protein epitopes were assessed with a novel Luminex-based peptide assay (LPA). Using machine learning techniques, a model was developed to predict different degrees of CMA. RESULTS: The baked milk reactive patients demonstrated the highest degree of IgE epitope binding, which was followed sequentially by fermented milk reactive, whole milk reactive, and outgrown. Data were randomly divided into two groups with 75% of the data utilized for model development (n = 68) and 25% for testing (n = 21). All 68 children used for training were correctly classified with models using IgE and IgG4 epitopes. The average cross-validation accuracy was much higher for models using IgE plus IgG4 epitopes by LPA (84.8%), twice the performance of the serum component proteins assayed by UniCAP (41.9%). The performance of the model on "unseen data" was tested using the 21 withheld patients, and the accuracy of IgE was 86% (AUC = 0.89) while of IgE+IgG4 model was 81% (AUC = 0.94). CONCLUSION: Using a novel high-throughput LPA, we were able to distinguish the diversity of IgE/IgG4 binding to epitopes in the varying CMA phenotypes. LPA is a promising tool to predict correctly different degrees of CMA.

The nasal microbiome in asthma.

BACKGROUND: Nasal microbiota may influence asthma pathobiology. OBJECTIVE: We sought to characterize the nasal microbiome of subjects with exacerbated asthma, nonexacerbated asthma, and healthy controls to identify nasal microbiota associated with asthma activity. METHODS: We performed 16S ribosomal RNA sequencing on nasal swabs obtained from 72 primarily adult subjects with exacerbated asthma (n = 20), nonexacerbated asthma (n = 31), and healthy controls (n = 21). Analyses were performed using Quantitative Insights into Microbial (QIIME); linear discriminant analysis effect size (LEfSe); Phylogenetic Investigation of Communities by Reconstruction of Unobserved States; and Statistical Analysis of Metagenomic Profiles (PICRUSt); and Statistical Analysis of Metagenomic Profiles (STAMP). Species found to be associated with asthma activity were validated using quantitative PCR. Metabolic pathways associated with differentially abundant nasal taxa were inferred through metagenomic functional prediction. RESULTS: Nasal bacterial composition significantly differed among subjects with exacerbated asthma, nonexacerbated asthma, and healthy controls (permutational multivariate ANOVA, P = 2.2 × 10-2). Relative to controls, the nasal microbiota of subjects with asthma were enriched with taxa from Bacteroidetes (Wilcoxon-Mann-Whitney, r = 0.33, P = 5.1 × 10-3) and Proteobacteria (r = 0.29, P = 1.4 × 10-2). Four species were differentially abundant based on asthma status after correction for multiple comparisons: Prevotella buccalis, Padj = 1.0 × 10-2; Dialister invisus, Padj = 9.1 × 10-3; Gardnerella vaginalis, Padj = 2.8 × 10-3; Alkanindiges hongkongensis, Padj = 2.6 × 10-3. These phyla and species were also differentially abundant based on asthma activity (exacerbated asthma vs nonexacerbated asthma vs controls). Quantitative PCR confirmed species overrepresentation in asthma relative to controls for Prevotella buccalis (fold change = 130, P = 2.1 × 10-4) and Gardnerella vaginalis (fold change = 160, P = 6.8 × 10-4). Metagenomic inference revealed differential glycerolipid metabolism (Kruskal-Wallis, P = 1.9 × 10-4) based on asthma activity. CONCLUSIONS: Nasal microbiome composition differs in subjects with exacerbated asthma, nonexacerbated asthma, and healthy controls. The identified nasal taxa could be further investigated for potential mechanistic roles in asthma and as possible biomarkers of asthma activity.

What Characteristics Confer Proteins the Ability to Induce Allergic Responses? IgE Epitope Mapping and Comparison of the Structure of Soybean 2S Albumins and Ara h 2.

Ara h 2, a peanut 2S albumin, is associated with severe allergic reactions, but a homologous protein, soybean 2S albumin, is not recognized as an important allergen. Structural difference between these proteins might explain this clinical discrepancy. Therefore, we mapped sequential epitopes and compared the structure of Ara h 2, Soy Al 1, and Soy Al 3 (Gly m 8) to confirm whether structural differences account for the discrepancy in clinical responses to these two proteins. Commercially synthesized peptides covering the full length of Ara h 2 and two soybean 2S albumins were analyzed by peptide microarray. Sera from 10 patients with peanut and soybean allergies and seven non-atopic controls were examined. The majority of epitopes in Ara h 2 identified by microarray are consistent with those identified previously. Several regions in the 2S albumins are weakly recognized by individual sera from different patients. A comparison of allergenic epitopes on peanut and soybean proteins suggests that loop-helix type secondary structures and some amino acids with a large side chain including lone electron pair, such as arginine, glutamine, and tyrosine, makes the peptides highly recognizable by the immune system. By utilizing the peptide microarray assay, we mapped IgE epitopes of Ara h 2 and two soybean 2S albumins. The use of peptide microarray mapping and analysis of the epitope characteristics may provide critical information to access the allergenicity of food proteins.

CD23+IgG1+ memory B cells are poised to switch to pathogenic IgE production in food allergy.

Food allergy is caused by allergen-specific immunoglobulin E (IgE) antibodies, but little is known about the B cell memory of persistent IgE responses. Here, we describe, in human pediatric peanut allergy, a population of CD23+IgG1+ memory B cells arising in type 2 immune responses that contain high-affinity peanut-specific clones and generate IgE-producing cells upon activation. The frequency of CD23+IgG1+ memory B cells correlated with circulating concentrations of IgE in children with peanut allergy. A corresponding population of "type 2-marked" IgG1+ memory B cells was identified in single-cell RNA sequencing experiments. These cells differentially expressed interleukin-4 (IL-4)- and IL-13-regulated genes, such as FCER2/CD23+, IL4R, and germline IGHE, and carried highly mutated B cell receptors (BCRs). In children with high concentrations of serum peanut-specific IgE, high-affinity B cells that bind the main peanut allergen Ara h 2 mapped to the population of "type 2-marked" IgG1+ memory B cells and included clones with convergent BCRs across different individuals. Our findings indicate that CD23+IgG1+ memory B cells transcribing germline IGHE are a unique memory population containing precursors of high-affinity pathogenic IgE-producing cells that are likely to be involved in the long-term persistence of peanut allergy.

Integrated study of systemic and local airway transcriptomes in asthma reveals causal mediation of systemic effects by airway key drivers.

BACKGROUND: Systemic and local profiles have each been associated with asthma, but parsing causal relationships between system-wide and airway-specific processes can be challenging. We sought to investigate systemic and airway processes in asthma and their causal relationships. METHODS: Three hundred forty-one participants with persistent asthma and non-asthmatic controls were recruited and underwent peripheral blood mononuclear cell (PBMC) collection and nasal brushing. Transcriptome-wide RNA sequencing of the PBMC and nasal samples and a series of analyses were then performed using a discovery and independent test set approach at each step to ensure rigor. Analytic steps included differential expression analyses, coexpression and probabilistic causal (Bayesian) network constructions, key driver analyses, and causal mediation models. RESULTS: Among the 341 participants, the median age was 13 years (IQR = 10-16), 164 (48%) were female, and 200 (58.7%) had persistent asthma with mean Asthma Control Test (ACT) score 16.6 (SD = 4.2). PBMC genes associated with asthma were enriched in co-expression modules for NK cell-mediated cytotoxicity (fold enrichment = 4.5, FDR = 6.47 × 10-32) and interleukin production (fold enrichment = 2.0, FDR = 1.01 × 10-15). Probabilistic causal network and key driver analyses identified NK cell granule protein (NKG7, fold change = 22.7, FDR = 1.02 × 10-31) and perforin (PRF1, fold change = 14.9, FDR = 1.31 × 10-22) as key drivers predicted to causally regulate PBMC asthma modules. Nasal genes associated with asthma were enriched in the tricarboxylic acid (TCA) cycle module (fold enrichment = 7.5 FDR = 5.09 × 10-107), with network analyses identifying G3BP stress granule assembly factor 1 (G3BP1, fold change = 9.1 FDR = 2.77 × 10-5) and InaD-like protein (INADL, fold change = 5.3 FDR = 2.98 × 10-9) as nasal key drivers. Causal mediation analyses revealed that associations between PBMC key drivers and asthma are causally mediated by nasal key drivers (FDR = 0.0076 to 0.015). CONCLUSIONS: Integrated study of the systemic and airway transcriptomes in a well-phenotyped asthma cohort identified causal key drivers of asthma among PBMC and nasal transcripts. Associations between PBMC key drivers and asthma are causally mediated by nasal key drivers.