Allergic reactions occur when IgE molecules become crosslinked by antigens such as food proteins. Here we create the ‘AllerScan’ programmable phage display system to characterize the binding specificities of anti-allergen IgG and IgE antibodies in serum against thousands of allergenic proteins from hundreds of organisms at peptide resolution. Using AllerScan, we identify robust anti-wheat IgE reactivities in wheat allergic individuals but not in wheat-sensitized individuals. Meanwhile, a key wheat epitope in alpha purothionin elicits dominant IgE responses among allergic patients, and frequent IgG responses among sensitized and non-allergic patients. A double-blind, placebo-controlled trial shows that alpha purothionin reactivity, among others, is strongly modulated by oral immunotherapy in tolerized individuals. AllerScan may thus serve as a high-throughput platform for unbiased analysis of anti-allergen antibody specificities. A diverse array of antigens can trigger allergic reactions. Here the authors present the ‘AllerScan’ programmable phage display library, which is an efficient and unbiased approach for profiling anti-allergen antibody reactivities at cohort scale, with which a key wheat epitope is found to distinguish between wheat allergy and tolerance. In patients with IgE-mediated food allergy, exposure to allergenic food results in cross-linking of pre-formed food-specific IgE (fs-IgE) bound to the high-affinity IgE receptor FcεRI on the surface of mast cells and basophils, causing potentially life-threatening allergic reactions. The high prevalence of food allergy has led to an ever-increasing need for food allergy testing in clinical practice. While oral food challenge is the gold standard for diagnosing food allergy, this procedure is time-consuming, requires highly trained personnel, and can cause an acute allergic reaction. Therefore, the diagnosis of food allergy is often based on a combination of patient clinical history and the results of fs-IgE testing and skin prick testing. These tests, however, often detect sensitization to foods that are not associated with symptoms upon ingestion, which can lead to unnecessary food avoidance. The shortcomings of fs-IgE testing are exemplified in the diagnosis of IgE-mediated wheat allergy, where a meta-analysis found that wheat-specific IgE levels have a specificity of only 43% in predicting wheat allergy2. Strong cross-reactivity between grass pollen and wheat is likely a contributing factor to the high rate of false-positive tests3.In recent decades, food allergy has emerged as a major public health issue, affecting up to 10% of the population in westernized countries5. These approaches have revealed that certain immunodominant peptides, as well as overall greater diversity of IgE epitopes recognized, are associated with more severe reactions and a greater likelihood of having persistent allergy in patients with milk and egg allergy4.While component testing has emerged as a useful adjunct for diagnosing allergy for select foods , this approach is limited to testing single allergens. Comprehensive characterization of allergies via component testing requires relatively large sample volumes and significant expense. Other strategies to refine the diagnosis of food allergy have relied on epitope mapping, which evaluates IgE binding to a library of contiguous short peptides that compose allergenic proteins. Several methodologies have been developed, including SPOT membranes, microarray based immunoassays, and most recently Bead-Based Epitope AssaysHowever, these approaches are largely only capable of assessing antibody reactivities to a select number of known allergenic components  and are frequently difficult to interpret. Importantly, the identification of novel allergenic epitopes is both costly and time-intensive; inexpensive high-throughput approaches that efficiently identify novel epitopes would therefore have great utility to inform clinical component and allergenic epitope test development.8 we use oligonucleotide library synthesis to encode a database of allergenic peptide sequences for display on T7 bacteriophages (the “AllerScan” library), which can be analyzed using high-throughput DNA sequencing. While lacking in highly conformational, discontinuous, and post-translationally modified epitopes, PhIP-Seq enables high-resolution analysis of longer, higher quality peptides than is otherwise possible with synthetic peptide microarrays, and at a dramatically reduced per-sample cost.In this work, we present a programmable phage display based method to comprehensively analyze anti-allergen IgE and IgG antibodies to 1847 allergenic proteins, tiled with overlapping 56 amino acid peptides, in a single multiplex reaction. Similar to other Phage ImmunoPrecipitation Sequencing (PhIP-Seq) librariesUsing the AllerScan phage display library, we identify thousands of IgE and IgG antibody reactivities against hundreds of distinct organisms with peptide level resolution. We identify robust differences in the anti-wheat IgE reactivities of wheat allergic individuals compared to both non-allergic and wheat-sensitized individuals. Finally, we identify a wheat allergen epitope in alpha purothionin that may prove to be particularly useful in distinguishing wheat allergy from wheat sensitization. AllerScan is a massively multiplexed serological assay with utility for correlating clinical characteristics with anti-allergen IgE and IgG reactivity at cohort scale.9 was downloaded from UniProt (accessed 6 August 2017) and used as input to the PhIP-Seq pepsyn library design pipeline7. The 1847 proteins of the Allergome database were represented as a set of 19,332 56 amino acid peptide tiles with 28 amino acid overlaps, which were encoded by a library of synthetic 200-mer oligonucleotides , individuals with wheat “sensitivity”13 , and non-allergics . An expansive set of 577 wheat peptides was found to be commonly IgE reactive in the wheat allergic group out of a total 1525 wheat-associated peptides in the AllerScan library. Minimal reactivity was detected to the phage-displayed wheat peptides in sera from either sensitized or non-allergic individuals . Only in the IgG immunocaptures were the breadths of responses to wheat peptides significantly higher in the sensitized versus the non-allergic individuals (p = 0.0034). Taken together, these findings suggest that the breadth of the anti-wheat peptide IgE repertoire may have utility in distinguishing between wheat allergy and sensitization.Among wheat allergic IgE responses, we noted both extensive immunodominance and distinct patterns of reactivity. Upon examination of IgG reactivity to wheat peptides among the same three groups, we noted dramatic elevation of reactivity among allergic individuals—largely to the same IgE immunodominant epitopes, along with detectable but relatively less reactivity among the sensitized individuals, and minimal reactivity among the non-allergic individuals Fig. . Severality Fig.  versus n19. The N-terminal region contains a dominant epitope , and after 1 year of active wheat OIT. In these AllerScan analyses, data are plotted in a pairwise fashion; unchanged reactivities fall along the y = x diagonal, whereas reactivities that increase or decrease over time appear in the upper left or lower right quadrant, respectively. Most individuals in the placebo arm of the trial exhibited minimal changes to their anti-allergen IgE or IgG profiles . Twenty-three participants in the previously described food allergy cohort were also enrolled in a randomized, double-blind, placebo-controlled clinical trial of WOITsee Fig. . Overallp = 0.01, Fig. p = 0.0006, Fig. During the placebo phase of the trial, neither anti-wheat breadth nor specific reactivity to alpha purothionin changed significantly, either in IgE or IgG Fig. . In contWe have created the AllerScan phage display library, which is composed of all protein sequences present in the Allergome database and used it to characterize both IgE and IgG antibody reactivities in a cohort of food allergic individuals. Similar to previous PhIP-Seq libraries, AllerScan employs high-throughput oligonucleotide synthesis to efficiently encode high quality, overlapping 56 amino acid peptide sequences. Currently, polypeptides of this length cannot be reliably synthesized chemically. Here, we used IgE-specific immunoprecipitation of phage-displayed peptides, followed by high-throughput DNA sequencing to identify reactive peptides. Incorporation of liquid-handling automation, as we have done in this study, increases sample throughput and enhances assay reproducibility. High levels of sample multiplexing can be achieved by incorporating DNA barcodes into the PCR amplicon, prior to pooling and sequencing; at a sequencing depth of ~10-fold, ~500 AllerScan assays can be analyzed on a single run of an Illumina NextSeq 500. Batched analyses can therefore bring down the assay cost to just a few dollars per sample.8, will enable a broader analysis of IgE reactivity.PhIP-Seq with the AllerScan library provides quantitative antibody reactivity data at epitope level for about 2000 proteins derived from hundreds of organisms. This unbiased approach evaluates antibody reactivities to major and minor allergens, including both well-defined and poorly studied epitopes. The library can be readily updated by simply adding new allergens as they are identified. Future studies using additional libraries, such as the human or VirScan phage libraries21, protein denaturation during digestion is thought to reduce their functional significance for food allergies22. It is therefore possible that the AllerScan library may exhibit reduced sensitivity for non-food allergic antibodies. This topic will be addressed in future work.Limitations of all programmable phage-based assays include the lack of post- translational modifications and discontinuous epitopes. While these two types of epitopes are likely critical for certain allergensIn this study we utilized a constant input of total IgE, versus sIgE, to explore the unbiased utility of AllerScan for identifying any potential allergic antibody reactivity. Normalizing by sIgE input may be useful in future studies focused on specific allergens. We wondered, however, whether the amount of wheat sIgE confounded our analysis of allergic versus sensitized reactivities. Supplementary Fig. 23. The diagnosis of IgE-mediated wheat allergy is particularly challenging as wheat-specific IgE levels do not reliably predict allergy. Although a number of major wheat allergens have been identified in patients with IgE-mediated wheat allergy, IgE component testing for these proteins has not reached the sensitivity and specificity necessary to incorporate them into clinical practice. We sought to characterize anti-wheat antibody responses due to the current gap in knowledge about their relevant fine specificities. Our results suggest that peptide-level IgE reactivity may enhance discrimination between wheat allergy and sensitization. We also found that sensitized  individuals harbored elevated anti-wheat IgG responses compared to their non-allergic counterparts. These data are consistent with the possibility that sensitized individuals also harbor IgE responses to wheat, but that compensatory IgG can effectively block pathogenic IgE binding to wheat epitopes24.Here we analyzed over one million antibody–allergen peptide interactions in a comprehensive study of pan-allergen serology from a cross-sectional cohort of patients with peanut and/or wheat allergies, as well as non-allergic controls. Peanut and wheat allergens were the most widely recognized in this cohort, but we also detected reactivities against many other allergen sources including tree nuts, invertebrate tropomyosin, and milk proteins. This observation is not unexpected since many of the participants in our study had additional food allergies, which is often the case in patients with food allergy in the general population25. Additionally, we compared isotype differences in wheat peptide reactivity and found notable discordance in IgE and IgG reactivity for most peptides, as has been observed in other epitope studies; only 16% of reactive peptides were bound by both IgE and IgG isotypes. It is possible that this discordance may be partially explained by competition between the two antibody classes. Future experiments in which purified IgE and IgG are used for AllerScan analysis will test this hypothesis. Future studies using AllerScan may also provide insight into questions about B cell development, class-switching, and affinity maturation, particularly when used in high-resolution longitudinal studies or in animal models of allergic disease.We observed consistent patterns of wheat reactivity among the allergic cohort; some peptides were reactive in over 80% of individuals with wheat allergy, whereas most peptides did not react with sera from any individual included in this study. Dominant wheat reactivities tended to occur in highly repetitive regions of allergenic proteins; this phenomenon has also been described for other allergens21. We used sequence alignment to characterize homology among all dominant epitopes, which revealed modest homology between all gliadin peptides. Reactive omega and gamma gliadin epitopes were particularly homologous, which has been previously reported26. IgE reactive LMW glutenin peptides also exhibited modest sequence homology with the gliadins, but did not share homology with HMW glutenin peptides. The reactive peptides derived from HMW glutenin were characterized by a small number of highly repetitive motifs. All reactive HMW glutenin peptides possessed at least one IgE-binding epitope with strong consensus to previously reported HMW glutenin epitopes21.In wheat allergic individuals, the most reactive wheat epitopes were derived from alpha, beta, gamma, and omega gliadin and both HMW and LMW glutenin . These proteins all harbor highly repetitive domains19. IgE that recognizes this protein has been associated with a fourfold increased risk of experiencing severe allergic reactions to wheat27. Interestingly, while we did not detect an association between allergy severity and alpha purothionin reactivity in our study, we did identify anti-alpha purothionin IgG reactivity as the most prevalent (72%) reactivity in non-allergic and sensitized individuals, whereas IgG reactivity was low among individuals with wheat allergy. Conversely, IgE reactivity to alpha purothionin was found in 28% of wheat allergic individuals and only 2% of non-allergic/sensitized individuals. IgG and IgE reactivity to alpha purothionin may therefore be quite useful in distinguishing between allergy and sensitization to wheat.Alpha purothionin is a plant defense protein abundantly expressed in wheat seeds28. Interestingly, most (82.6%) allergic patients receiving WOIT experienced an increase in IgG reactivity to alpha purothionin. We additionally compared changes in IgE and IgG wheat reactivity with clinical responses and found no stasticially significant associations after multiple hypothesis test correction; a future, more sufficiently powered, study may identity significant correlations.The quantitative nature of AllerScan data enabled a longitudinal analysis of response to WOIT in a placebo-controlled, double-blind study involving 23 wheat allergic individuals. In response to WOIT, we noted a dramatic shift from IgE to IgG reactivity with wheat peptides, whereas reactivity to other allergens was not affected. As expected, individuals experienced no change in anti-wheat reactivity while in the placebo arm of the trial. Nearly every participant receiving treatment exhibited an overall reduction in anti-wheat IgE repertoire breadth and a concurrent increase in IgG repertoire breadth, an observation that has been reported in other food oral immunotherapy trialsIn conclusion, AllerScan is a approach for epitope-level characterization of allergen-associated IgE and IgG responses at cohort scale. We have demonstrated its utility for broad pan-allergen analysis as well as for characterizing the fine specificities of anti-wheat antibodies. Our initial research has confirmed numerous previously known properties of wheat allergy and has revealed reactivity towards an epitope that discriminates between wheat allergy and sensitization. AllerScan is therefore a valuable tool for allergy research and may ultimately provide a powerful adjunct to current methods for diagnosing food allergy and monitoring response to immunotherapy.Sera for AllerScan were obtained from 58 patients with IgE-mediated food allergy along with 25 age-matched healthy controls who were enrolled on a Natural History of Food Allergy protocol at the National Institutes of Health (NIH). Clinical metadata on the participants are shown in Supplementary Table 20. Briefly, participants with wheat allergy confirmed by a positive double-blind, placebo-controlled oral food challenge (DBPCFC) to wheat at baseline were randomized 1:1 to OIT with vital wheat gluten or placebo. Participants underwent dose escalation every 2 weeks until they reached a daily maintenance dose of 1445 mg of wheat or placebo protein. After approximately 1 year of treatment (minimum of 8 weeks of maintenance dosing), participants underwent a DBPCFC to wheat (cumulative dose of 7443 mg wheat protein) to evaluate for desensitization and were subsequently unblinded. Participants in the active arm continued on active wheat OIT, while the placebo arm crossed over to active treatment (maximum maintenance dose of 2748 mg wheat protein) for approximately 1 year. Individuals evaluated by AllerScan were enrolled in the WOIT trial at Johns Hopkins University and the Icahn School of Medicine at Mount Sinai in New York. The WOIT trial was approved by both institutional review boards and informed consent was obtained from all participants or their parents. All primary outcomes of the trial were published at clinicaltrials.gov (NCT01980992).Sera were also evaluated from 23 wheat allergic individuals who participated in a randomized, double-blind, placebo-controlled WOIT trial. Details of the clinical study are described in ref. 7 Briefly, we downloaded all protein sequences from Uniprot database included in the Allergome and collapsed on 90% sequence identity [https://www.uniprot.org/uniref/?query=uniprot:+identity:0.9]. The UniProt clustering algorithm returned representative sequences for each protein cluster. We designed peptide sequences 56 amino acids in length that overlap by 28, to tile through all representative allergen proteins. Next, we reverse-translated these sequences into DNA nucleotide sequences optimized for Escherichia coli expression and added adapter sequences to both the 5′ and 3′ ends. These 200 nucleotide sequences were synthesized by Twist Bioscience. This oligonucleotide library was PCR amplified and then restriction cloned into the T7FNS2 vector backbone. The resulting library was packaged via the T7 Select Packaging Kit (EMD Millipore) and then expanded for use as a PhIP-Seq library.The AllerScan library was constructed following the previously detailed PhIP-Seq protocol8. Briefly, an IgG-specific enzyme-linked immunosorbent assay was used to quantify serum concentrations (Southern Biotech). Two micrograms of IgG was added to 1 mL of AllerScan library at 1.9 × 109 pfu for each reaction. Serum and phage library were rotated overnight at 4 °C, after which 20 μL of protein A and 20 μL of protein G-coated magnetic beads  were added to each reaction, which was rotated an additional 4 h at 4 °C. Beads were subsequently washed three times in 0.1% NP-40 and then resuspended in a Herculase II Polymerase (Agilent cat # 600679) PCR master mix. These PCR reactions underwent 20 cycles of PCR. Two microliters of each reaction was added to a new PCR reaction containing sample-specific barcoding primers. These reactions underwent an additional 20 cycles of PCR. The final amplified product was pooled and sequenced using an Illumina NovaSeq 6000 or NextSeq 500.IgG screening of the AllerScan library was performed as described previously9 pfu for each reaction and 10 μL of omalizumab-coated M280s was used for each immunoprecipitation. All other steps for IgE PhIP-Seq were identical to the IgG PhIP-Seq methodology described above.For IgE Phip-Seq, biotinylated omalizumab (Labome—KBI1021) was conjugated to Streptavidin-bound M280 magnetic beads (ThermoFisher—11205D) at 4× binding capacity. Excess unbound omalizumab was washed away with 0.01% PBST. One hundred nanograms of IgE was added to 1 mL of AllerScan library at 1.9 × 10Sequencing reads were mapped to the AllerScan library, requiring perfect matches. We counted the number of times a clone was detected in each immunoprecipitate, creating a read count matrix. Next, we utilized the “edgeR” R software package, which compares the signal detected in each sample against a set of negative control “mock” immunoprecipitations that were performed without serum, using a negative binomial model, returning both a test statistic and fold-change value for each peptide in every sample, thus creating enrichment and fold-change matrices.p values, and fold changes of at least 100, 0.001, and 5, respectively. Hits fold-change matrices report the fold-change value for “hits” and “1” for peptides that are not hits.Significantly enriched peptides, called “hits”, required counts, N = 23), we used a lenient threshold for response classification: requiring an increase of >1000 mg of wheat extract between baseline and after treatment to be considered a response.For the specific analysis of WOIT trial data (This study compared reactivities from all placebo patients before and after placebo treatment and compared reactivities from all patients before and after 1 year of active treatment. Two patients did not complete the trial, two patients failed to respond by this metric, and the remaining 19 patients were considered responders.All analyses were developed and implemented in R 3.6.1. All heatmaps were constructed using the pheatmap package and all network graph analyses utilized the igraph software package; all other plots were built using the tidyverse package suite. Heatmap clustering used the Euclidean distance measure and the Ward.D2 linkage measure. All statistical tests were two-sided Wilcoxon signed-rank tests unless otherwise specified.Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Evidence was found for the existence of a highly viscous phase of water that exists between 180 and 220 K based on the observation that water can leave the intermembrane space at these low temperatures. Similar measurements are described in the literature for purple membrane (PM) samples. From a comparison with results from this natural membrane by using the same flash-cooling protocol, it is found that in the case of pure lipid samples, less water is trapped and the water flows out at lower temperatures. This suggests that the water is less hindered in its movements than in the PM case. It is shown that at least the Lβ′-phase of DMPC can be trapped likely by flash cooling; upon heating to about 260 K, it transforms to another phase that was not fully characterized.Neutron diffraction was used to study the behavior of water present in phospholipid multilamellar stacks from 1,2-dimyristoyl- The lipid presents a gel-to-gel pre-transition (Lβ′ > Pβ′) at 284.15 K and a gel-to-liquid crystalline main acyl chain crystallization transition (Pβ′ > Lα) at 296.5 K upon temperature increase  with two molecules in the asymmetric unit.1,2-Dimyristoyl-A study of the literature on phospholipid phases at low temperatures indicates a strong dependence on the conditions of hydration and temperature of equilibration of the bilayers so that various phases have been identified like the Pcc phase  appearing in DMPC and DPPC samples, when hydration and storage occur at 4°C , used without further purification. In 6 ml trifluoroethanol and 2 ml chloroform was dissolved 400 mg of the powder, so that a clear homogenous solution was obtained, which was kept overnight in a freezer at −20°C. The solution was carefully placed into the base of a 4 × 3 cm3He detector with a resolution of 2 mm in the horizontal and vertical directions. With the detector placed at 1 m from the sample, this corresponds to a resolution of 0.115°  and an angle of 14.7° for the whole detector. Measurements were carried out with a detector–sample distance of 1 m. To ensure that all measurements on one sample were made in the same orientation, the samples were aligned using the rocking-curve method, every time the sample was remounted, for example, after each flash cooling. First, the sample is placed approximately orthogonal to the neutron beam. Then a simple ω-scan, where ω is the sample angle from ω = 70° to 110°, is carried out by observing the intensity of the first lamellar peak, at a detector angle of γ = 5°, corresponding to the lamellar spacing d of the membrane stack. This curve is ideally reminiscent of a Gaussian broken by two characteristic minima due to high absorption where the sample is oriented parallel to the incident or diffracted beam. We choose the first minimum to set ω = 90° and measured the position of the lamellar peak at ω = 92°, where the intensity of the rocking curve is maximum. The 2D images were integrated and plotted vs. Q, the scattering vector perpendicular to the membrane stacks [Q = (4π/λ)sinθ with 2θ being the scattering angle]. Temperature control was carried out in an “Orange” ILL Cryostat on D16  and ice formation  reflection of hexagonal ice; called ice peak 1 hereafter] and 3.67 Å [Q = 1.71 Å−1; originating from amorphous ice, the (1 1 1) reflection of cubic ice, or the (0 0 2) reflection of hexagonal ice; called ice peak 2 hereafter; Q of the lipid peak , the evolution of d globally follows the one during the preceding SHFC protocol down to 265 K and then continues to decrease to reach a d value of 48.7 Å at 100 K. During subsequent heating (SH protocol) to 300 K, d evolves almost identically to the preceding SC protocol with the exception of a hysteric behavior between 270 and 280 K.Upon heating (SHFC protocol) to 180 K, ice peak 1 and the presence of ice peak 2 indicate the absence of hexagonal and the presence of cubic and/or amorphous ice, respectively. Upon heating (SHFC protocol), the integrated intensity of ice peak 2 remains more or less constant up to 190 K , ice peak 1 appears and increases in intensity at a high rate between 270 and 250 K and at a lower rate between 250 and 220 K and then fluctuates upon further cooling to 100 K. Ice peak 2 appears and increases in intensity in parallel to ice peak 1 down to 245 K and then slightly decreases in intensity during further cooling to 100 K. When comparing results from the SHFC and the SC protocols, it is evident that ice peak 1 intensities are higher than ice peak 2 intensities during cooling and lower during heating.After flash cooling, at 100 K, the absence of to 190 K . BetweenQ values during heating from 100 to 240 K  were attributed to the Lβ-phase of the lipid  and to td-spacing. For 100 K after SC, we have about 5.7 Å, which corresponds to roughly two layers of water   that is 20 K lower than in PM (200 K), suggests that the interlamellar water is less hindered in its movements by the pure-lipid bilayers than by the PM. Water flowing out of the intermembrane space above 180 K  and trapped lipids (260 K) gain mobility are different, in line with observations in single supported DMPC bilayers that suggested a decoupling of hydration water and membrane freezing , where C is a scale factor and Λ the hydration repulsion decay length  reflection of cubic ice is of a higher order than the (1 0 0) reflection from hexagonal ice. This might explain why the intensity of ice peak 2 in SHFC is much lower than that of ice peak 1 in SC, even though both peaks represent about the same amount of ice.In the SHFC experiment, the sample is in a metastable state after flash cooling. The water in the flash-cooled system did not form hexagonal ice. From the fact that the intensity of We studied the behavior of a bilayer stack of synthetic phospholipids (DMPC) in a hydrated environment, at cryo-temperatures, and present evidence for the existence of a highly viscous phase of water above 180 K in flash-cooled samples. In comparison with PMs (Weik et al., All datasets generated for this study are included in the article/supplementary material.JM, GF, and MW designed and performed the experiments. JM and MW did the data analysis. GF, GZ, and MW wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Few studies have focused on quantitatively analyzing nutrients from infant diets, compromising complementary feeding evaluation and health promotion worldwide.This study aimed to describe dietary intake in infants from 9 to 24 mo of age, determining nutrient intakes associated with the risk of underweight, wasting, and stunting.n = 169), Peru (n = 199), South Africa (n = 221), Tanzania (n = 210), Bangladesh (n = 208), India (n = 227), and Nepal (n = 229), totaling 1463 children and 22,282 food recalls. Intakes were corrected for within- and between-person variance and energy intake. Multivariable regression models were constructed to determine nutrient intakes associated with the development of underweight, wasting, and stunting at 12, 18, and 24 mo of age.Usual nutrient intakes from complementary feeding were determined by 24-h recalls collected when infants were 9–24 mo of age in communities from 7 low- and middle-income countries: Brazil : 0.90; 95% CI: 0.84, 0.96] and 24 mo , and wasting at 18  and 24 mo . Higher zinc intakes decreased the risk of underweight  and wasting  at 12 mo, and wasting  at 24 mo.Higher intakes of energy and zinc in complementary feeding were associated with decreased risk of undernutrition in the studied children. Data suggest these are characteristics to be improved in children's complementary feeding across countries. The timThe risk of undernutrition during the first 2 y of life increases during the complementary feeding period , 6. InsuAlthough breastfeeding practices have been evaluated continuously and promoted through research and policies, complementary feeding evaluation and thus promotion have encountered several limitations. The WHO core indicators , standarThe MAL-ED  study is a longitudinal birth cohort study in 8 low- or middle-income countries . In thisThe study was approved by the Institutional Review Board for Health Sciences Research, University of Virginia, USA as well as the respective governmental, local institutional, and collaborating institutional ethical review boards at each site: Committee for Ethics in Research, Universidade Federal do Ceará; National Ethical Research Committee, Health Ministry, Council of National Health in Brasília and Fortaleza, Brazil (Brazilian site); Institutional Review Board, Johns Hopkins University, in Baltimore, MD, USA; PRISMA Ethics Committee; Health Ministry, in Loreto, Peru (Peruvian site); Health, Safety and Research Ethics Committee, University of Venda; Department of Health and Social Development, Limpopo Provincial Government, in Venda, South Africa (South African site); Medical Research Coordinating Committee, National Institute for Medical Research; Chief Medical Officer, Ministry of Health and Social Welfare in Haydom, Tanzania (Tanzanian site); Ethical Review Committee, icddr,b in Dhaka, Bangladesh (Bangladesh site); Institutional Review Board, Christian Medical College in Vellore, India and the Health Ministry Screening Committee, Indian Council of Medical Research (Indian site); and Institutional Review Board, Institute of Medicine, Tribhuvan University; Ethical Review Board, Nepal Health Research Council; Institutional Review Board, Walter Reed Army Institute of Research in Bhaktapur, Nepal . Informed written consent was obtained from the parent or legal guardian of each participating child.n = 169); Loreto, Iquitos, Peru (PEL) (n = 199); Venda, South Africa (SAV) (n = 221); Haydom, Tanzania (TZH) (n = 210); Dhaka, Bangladesh (BGD) (n = 208); Vellore, India (INV) (n = 227); and Bhaktapur, Nepal (NEB) (n = 229). These communities were in urban , peri-urban (South Africa), and rural contexts (Peru and Tanzania). Although Naushahro Feroze, Pakistan (PKN) was part of the MAL-ED, data from this site are not used here owing to measurement quality concerns   for both weights and lengths were >0.9, and all quality control procedures are described by Richard et al.  the child's consumption in the previous 24 h of breast milk, animal milk, formula, other liquids, water, tea, fruit juice, semisolids, and specific solid foods . BreastfBeginning at 9 mo and monthly thereafter, the caregiver was asked to recall non-breast-milk food intake over the prior 24 h to estimate the energy and nutrient intake of the child using the 24-h recall method. These dietary data were collected monthly (starting at 9 mo) by trained fieldworkers, using forms completed by hand, and included details on the ingredients and preparation steps  for complex local preparations.The sites used the same 24-h recall form for data collection. Training for using the 24-h recall technique was conducted by the Nutrition Technical Subcommittee for each study site following general principles . Each siData were double-entered into a computer and transmitted to the Data Coordinating Center, Bethesda, MD. Multiple searches to identify errors and retraining of field staff based on error identification were performed with researchers from the Johns Hopkins University Bloomberg School of Public Health (JHSPH). Communication with the researchers at JHSPH resolved issues or questions identified during data collection. Then, energy and nutrient analyses were performed at JHSPH, using site-specific food composition tables created for the study in Excel 2013 (Microsoft) , 20, 21.Campylobacter spp., Shigella, enteroaggregative Escherichia coli(EAEC), typical enteropathogenic Escherichia coli(tEPEC), atypical enteropathogenic Escherichia coli (aEPEC), enterotoxigenic Escherichia coli (ETEC), Shiga toxin-producing Escherichia coli (STEC), Aeromonas, Helicobacter pylori, Plesiomonas, Salmonella, and Vibrio cholerae), viruses , and parasites  was performed to return the estimated usual intakes to their original scales. In order to control for confounding factors inherent in total energy intake, adjustment of the nutrient intake by energy using the residual method was done . BrieflyU test was used to compare energy and nutrient intakes between the children with or without underweight, wasting, and stunting. The differences in intake between the children with or without underweight, wasting, and stunting were expressed as percentages considering children without underweight, wasting, and stunting as the reference value.Nutrient intakes are presented as median intakes and IQRs in the 3 time periods. The Mann–Whitney r > 0.7) were excluded from the adjusted models. Final model adjustment was observed through the Omnibus tests of model coefficients, with P values < 0.05 considered significant. The Hosmer–Lemeshow test was also used, considering P values > 0.05 as reliable. Adjusted odds ratios (AORs) and 95% CIs were shown to assess the risk association found between nutrient intake and the outcome analyzed in the model. The analysis was performed using SPSS version 23 (IBM).Logistic regressions were constructed first in bivariate analyses, exploring the effect of a single nutrient intake on the outcomes of underweight, wasting, and stunting at 12, 18, and 24 mo, and crude ORs and 95% CIs were calculated. Then, 3 multivariable logistic regression models were constructed for each time period, considering as outcomes the presence of underweight, wasting, or stunting at 12, 18, and 24 mo. Because infections compromise nutritional status , 28, bacn = 202 (14.0%), n = 76 (5.3%), and n = 384 (26.6%) of the studied children at 12 mo, respectively; n = 230 (15.9%), n = 68 (4.7%), and n = 545 (37.9%) of the children at 18 mo, respectively; and n = 257 (17.9%), n = 63 (4.4%), and n = 551 (38.4%) at 24 mo, respectively (P < 0.001).Underweight, wasting, and stunting were present in ectively . AlthougP = 0.005). Prevalences of underweight and stunting were not significantly associated with breastfeeding status in any of the studied periods.At 24 mo of age, 61.9% of children with wasting were partially breastfed  and 24 mo , and wasting at 18  and 24 mo . Higher zinc intakes were associated with decreased risk of underweight  and wasting  at 12 mo, and wasting  at 24 mo . Children from the South Asian sites  presented lower energy and zinc intakes than those from Latin American and African sites  and South Asian sites —zinc intakes from complementary feeding were lower and in the South Asian sites  these intakes tended to not increase over time. Also, in the South Asian sites, where energy intakes from complementary feeding were lower from 9 to 24 mo of age than at the other studied sites, undernutrition was more prevalent.In South Asia, <3 in 5 infants aged 6–8 mo consume soft, semisolid, or solid foods, indicating late initiation of complementary feeding. In this region, complementary foods for children aged 6–23 mo are primarily cereal‐based, lacking the essential growth‐promoting nutrients provided by fruits, vegetables, and foods of animal origin . These cOverall, children from the Latin American sites in the study (BRF and PEL) and those from Eastern and South Africa (TZH and SAV) presented similar energy intakes from complementary feeding. White et al.  found thChildren from BRF and SAV presented higher zinc intakes from complementary feeding than the other studied sites. In Brazil, as previously reported , these rBetter complementary feeding practices predict better linear growth outcomes, and our study reinforces that energy and zinc from complementary food are lower in children with stunting. For stunted children, we also found higher fiber intakes in the 3 time points evaluated. Zinc supplementation studies have shown this nutrient has a particularly positive effect on growth , and marNutrient intake recommendations may vary according to diet characteristics that determine bioavailability . In the One of the limitations of our results is that the data are not nationally representative in each country. Nevertheless, the findings from the present study may apply to other communities within the studied countries. The potential for type I errors could be a limitation considering the multiple nutrient exposures assessed across outcomes. We began to collect dietary recalls at 9 mo of age, although introduction of non-breast-milk foods began well before 6 mo for most study infants. Strengths of our study are the prospective data collection with 24-h dietary recalls, which allowed for the collection of 15–17 dietary intake recalls per child, with equivalent data collection procedures across the sites, and the use of local food composition tables. This is the first study, to the best of our knowledge, to use this kind of approach to understand complementary feeding and its associations with nutritional outcomes.Further studies should also explore pathways from direct host metabolism to effects on the microbiome or even possible pathogen virulence expression as they relate to energy and zinc intake associations with underweight, wasting, and stunting. Analyzing complementary feeding intake from a usual quantitative nutrient intake perspective is an advantage of the MAL-ED cohort study protocol. In addition, assessing nutrient intake at 3 different time points has allowed the observation of lower energy and zinc intakes in children with undernutrition during the first 2 y of life.In summary, these results show the importance of nutrient intake from complementary feeding for the prevention of undernutrition in terms of its 3 primary anthropometric outcomes. Higher energy and zinc intakes in complementary feeding were associated with decreased risk of undernutrition. Data suggest these are complementary feeding characteristics to be improved across sites. More research should be conducted to support governments to identify national constraints, and to design and to implement specific programs to improve complementary feeding.nxaa271_Supplemental_FileClick here for additional data file.


In addition to their role in mediating immediate hypersensitivity reactions, mast cells and basophils serve separate but critical functions as adjuvants for type 2 immunity in food allergy. Mast cells and basophils, activated by IgE, are key sources of IL-4 that tilts the immune balance away from tolerance and towards type 2 immunity by promoting the induction of Th2 cells along with the innate effectors of type 2 immunity, ILC2s, while suppressing the development of regulatory T cells and driving their subversion to a pathogenic pro-Th2 phenotype. This adjuvant effect of mast cells and basophils is suppressed when inhibitory signals are delivered by IgG antibodies signaling via FcγRIIb. This review summarizes current understanding of the immunoregulatory effects of mast cells and basophils and how these functions are modulated by IgE and IgG antibodies. Understanding these pathways could provide important insights into innovative strategies for preventing and/or reversing food allergy in patients.Food allergy is a major health issue, affecting the lives of 8% of U.S. children and their families. There is an urgent need to identify the environmental and endogenous signals that induce and sustain allergic responses to ingested allergens. Acute reactions to foods are triggered by the activation of mast cells and basophils, both of which release inflammatory mediators that lead to a range of clinical manifestations, including gastrointestinal, cutaneous, and respiratory reactions as well as systemic anaphylaxis. Both of these innate effector cell types express the high affinity IgE receptor, FcϵRI, on their surface and are armed for adaptive antigen recognition by very-tightly bound IgE antibodies which, when cross-linked by polyvalent allergen, trigger degranulation. These cells also express inhibitory receptors, including the IgG Fc receptor, FcγRIIb, that suppress their IgE-mediated activation. Recent studies have shown that natural resolution of food allergies is associated with increasing food-specific IgG levels. Furthermore, oral immunotherapy, the sequential administration of incrementally increasing doses of food allergen, is accompanied by the strong induction of allergen-specific IgG antibodies in both human subjects and murine models. These can deliver inhibitory signals  Global surveillance by the World Allergy Organization, shows that the prevalence of food allergies has been rising over the last decade in both developed and developing countries . This inFood allergies are hypersensitivity reactions that can be mediated by a wide range of humoral and cellular mechanisms. IgE-mediated food allergy is the most common and will be the focus of this review. It occurs in individuals who produce food-specific IgE antibodies. These subjects are often referred to as “sensitized”. These IgE antibodies are bound to the innate granulocytic effector cells of anaphylaxis, mast cells and basophils. Upon interaction with allergen and cell-bound IgE, the granule contents of these cells are released and, along with prostaglandin and leukotriene mediators rapidly produced by the same cells, act on a range of target tissues to trigger immediate physiologic responses . In the via the gut. However, many children experience adverse reactions following their initial ingestion of a food, suggesting alternate routes of immune priming. Emerging evidence suggests that sensitization can occur following cutaneous contact, especially in the setting of a disrupted skin barrier, as occurs in atopic dermatitis  in constraining both the emergence of food allergen-specific T helper cells and the production of allergen-specific IgE. However, in recent years, the ability of mast cells and basophils to exert adjuvant functions in immune sensitization to allergens and of IgG antibodies to block IgE-mediated food allergy has been recognized and the role of the inhibitory IgG receptor, FcγRIIb, in potently inhibiting food allergies has really come into focus. In the first part of this review, we briefly discussed the mechanisms, pathophysiology and key players in the disease. In the second part, we cover the evidence for a regulatory functions of mast cells, basophils, IgE and IgG and how they may be targeted clinically to counter food allergy.via sampling by antigen presenting cells (APCs)  . Oral ext manner –11. Thesto Tregs . A breakto Tregs , 14. TSLto Tregs . IL-33 pards Th2 , 17. IL-ards Th2 . By incrards Th2 . Researcards Th2 .via disrupted skin barrier has also been shown to promote mast cell expansion at a distance in the small intestine in a manner dependent on the induction of intestinal ILC2s by keratinocyte-derived IL-33 along with intestinal tuft cell-derived IL-25  along with the Vitamin D analogue MC903, Noti, and colleagues observed that TSLP produced by keratinocytes promotes basophil expansion and the induction of a Th2 response (driven by basophil-derived IL-4) with food allergy , 22. Muted IL-25 .via IL-4Rα induces MMC9 in a BATF-dependent mechanism, nicely linking the observed roles of IL-9 and IL-4 in mast cell homeostasis . IL-9 promotes mast cell growth and lack of IL-9 prevents the induction of food allergy in mouse models , 25. Traeostasis . IL-4 sieostasis . Investivia several routes, including sublingual (SLIT), epicutaneous (EPIT) and oral (OIT) (®) OIT is the only current FDA-approved treatment for immunotherapy. A major limitation of OIT is that the food unresponsive state that is achieved is transient and maintaining it requires continued regular ingestion of the food  . Peanut the food . Adjunctthe food , 34.+ hematopoietic progenitor cells. Mast cells are long-lived tissue resident cells that differentiate locally from bloodborne progenitors , emokines , 54.5 times less (100 ng/ml vs 10 mg/ml) than that of IgG. In addition to being present at such low concentrations, IgE has a notably short half-life, less than 1 day, while that of IgG is around 3 weeks (d 1x10-9mol/L). IgE effectively remains permanently attached to FcϵRI until internalized, which makes the tissue half-life of IgE to likely be on the order of weeks to months . An ITAM is a conserved sequence containing tyrosine separated from a leucine or isoleucine by two amino acids (YxxL/I). The ITAM tyrosine is a substrate for phosphorylation by signaling kinases such as Lyn and Syk. Two ITAMs are often found together separated by 6 to 8 amino acids (YxxL/I x(6-8) YxxL/I).There exist two isoforms of the high affinity IgE receptor, FcϵRI. The tetrameric αβγWhen multivalent antigens bind to FcϵRI-bound IgE, proximal FcϵRI aggregate into lipid rafts that are rich in cholesterol, sphingolipids, protein tyrosine kinases (PTKs), and GPI-anchored proteins. Recognition of one antigen molecule by Fab sites on two different IgE molecules bound to neighboring FcϵRI, results in receptor crosslinking and transphosphorylation of the ITAMs. Antigen recognition mediates a rapid cascade of signaling events that induce the activation of PTKs of the SRC and TEC families –59. ThisTyrosine residues on ITAMs on β and γ chains are phosphorylated by receptor-associated Lyn tyrosine kinase found constitutively associated with the β chain. Phosphorylation of tyrosine residues in the ITAMs provides docking sites for Src homology 2 (SH2) domain-containing kinases, such as Syk PTK to the γ chains and recruits additional Lyn PTK. Syk is phosphorylated by Lyn and activated by conformational changes , 62. PhoIn addition to driving the allergic effector functions of mast cells and basophils, there is evidence that some IgE antibodies can provide survival signals to mast cells even in the absence of allergen. These have been designated “cytokinergic” IgEs by Kawakami and colleagues . In the Glycosylation of IgE molecules has been shown to correlate with IgE binding to FcϵRI and for effective signaling upon receptor cross-linking. IgE antibodies are very heavily glycosylated when compared with other immunoglobulin isotypes. Anthony and colleagues demonstrated the functional significance of IgE glycans. They found that one at N394 was obligatory for FcϵRI binding . SubsequDer p 1. The released oligomeric CD23 heads are called soluble CD23 (sCD23) and retain their ability to bind IgE.IgE can also bind to and exert its effects through its low affinity receptor CD23 (FcϵRII). CD23 is broadly distributed with expression on B cells, dendritic cells, eosinophils, gastrointestinal and respiratory epithelial cells, and others . It is aIn addition to FcϵRI, human and mouse mast cells and basophils also express Fcγ receptors (FcγRs) that bind to IgG antibodies. FcγRs belong to the immunoglobulin superfamily and their patterns of expression vary among leukocytes –77. The i.v.-challenged mice lacking the Cϵ exons encoding the IgE heavy chain  and in a heat-labile reagin fraction , 80. Thevy chain , 81–83. vy chain , 85. Whivy chain . As demovy chain . The exiFcγRIIb is the only inhibitory IgG receptor and one of its main function is to turn off signals initiated by activating Fc receptors and the B cell receptor (BCR). The importance of IgG-mediated feedback control of humoral immunity by FcγRIIb has long been appreciated . Co-aggrUnlike activating receptors, the aggregation of FcγRIIb alone is of no consequence and binding of IgG or immune complexes solely to FcγRIIb does not inhibit the functions of a resting cell. The inhibitory receptor is expressed on the surface of cells in conjunction with activating receptors, and arming the inhibitory function of FcγRIIb requires an initial licensing signal by an activating receptor. When immunoglobulins on activating and inhibitory receptors recognize independent epitopes in cis on a common antigen, the receptors co-aggregate in lipid rafts for crosslinking to occur , 95. Fcγ3) to phosphatidylinositol diphosphate (PIP2)  see Figure 1strongly . Therefo2 portion of IgG was not capable of mediating this inhibitory effect, suggesting that the phenotype is dependent on receptor-mediated action of IgG.It is commonly understood that the inhibitory function of FcγRIIb is mediated by its coaggregation with an activating receptor by the same multivalent antigen. This is described as FcγRIIb-mediated “cis-inhibition”. However, Malbec et al. show that FcγRIIb can also mediate inhibition of activating receptors triggered independently of, and not co-aggregated with, FcγRIIb . The autDue to its known roles in inhibiting the activation of immune cells, FcγRIIb has been considered as a potential regulator in various immunological disorders, especially in autoimmune diseases.via activating FcγRs, and impaired FcγRIIb function has been shown to be involved in the disease pathogenesis (T232) have been shown to be more prevalent in areas where malaria is endemic and may confer protection against the infection (Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with autoantibody production affecting many organs including the skin, brain, joints, and kidneys. Abnormal B cell responses to immune complexes containing autoantigens is a feature of SLE. Immune complexes exert their effects ogenesis . In rheuogenesis . In a coogenesis . In a moogenesis . In contogenesis . For exaogenesis , 107. Vanfection .via FcϵRI, and IgG antibodies, sending ITIM-mediated inhibitory signals via FcγRIIb, are critical in controlling the acute effector functions of mast cells and basophils. These are the pathways that respectively drive immediate hypersensitivity reactions in food allergy, including anaphylaxis, and suppress acute reactions in subjects who have developed food-specific IgG responses. However, the opposing roles of IgE and IgG in food allergy extend significantly beyond these direct effects on effector cell activation, with immunoregulatory influences ranging from modulation of IgE receptor density and signaling thresholds in effector cells, to the IL-4-driven priming and maintenance of effective Th2 responses to food allergens and corresponding suppression and/or pathogenic reprogramming of Tregs. These immediate and downstream functions of food allergen-specific IgE and IgG antibodies extend the rationale for IgE blockade by omalizumab during OIT and form the mechanistic basis for the development of food allergen-specific IgG monoclonal antibodies as novel therapeutics for food allergy. Here we provide a detailed review of the immunomodulatory effects of IgE and IgG antibodies as they relate both to innate effector cell functions and to downstream regulation of adaptive immune responses in food allergy.The opposing effects of IgE antibodies, triggering activating input in vivo at physiologic IgE levels. IgE-/- mice had greatly reduced FcϵRI expression both on circulating basophils and on tissue mast cells, and both cell types rapidly upregulated FcϵRI following IgE infusion  and T cells important in driving type 2 inflammation , 122. Invia FcϵRI, mast cells positioned at mucosal surfaces and in the skin as well as basophils recruited to these sites can prime the allergic immune response by influencing both innate and adaptive immune cells. Effective induction of T cell responses to contact sensitizers applied to the skin occurs only when mast cells and IgE are present, and local cutaneous inflammatory responses to superantigens are mast cell dependent  both independently led to impaired Th2 responses to peanut ingestion and still permitted the development and expansion of Tregs. The observation that mast cell-deficient mice reconstituted with wild type, but not IL-4-deficient, mast cells had restored Th2 responses to peanut strongly implicated mast cells as a key source of IL-4 in this system. IL-4 also negatively impacts the numbers and functions of Tregs so that, while promoting Th2 immunity, it also suppresses and potentially subverts the mechanisms that keep it in check. Chatila and colleagues showed that IL-4 can subvert Tregs to a pathogenic phenotype, expressing Th2 transcription factor GATA-3 as well as IL-4, a state which contributes to, rather than suppresses, allergic disease  or basophil-deleted Bas-TRECK mice, Kawakami and colleagues demonstrated attenuation of clinical scores, diarrhea incidence and plasma levels of the mast cell protease mMCP-1 following intragastric allergen instillation in mice primed intraperitoneally and then enterally challenged with OVA . Total aIn addition to mast cells and basophils, ILC2s have been identified as critical innate immune sources of IL-4 and inducers of Type 2 immune responses. It turns out there is a critical interplay between these cell types, revealed in murine models of food allergy. Like Th2 cells, ILC2s express the transcription factor GATA-3 and secrete Th2 cytokines, including IL-5 and IL-13. Unlike Th2 cells, they lack T cell receptor (TCR) and cannot recognize antigen. In response to epithelial-derived cytokines, such as IL-25 and IL-33, they produce large amounts of IL-5, IL-9, and IL-13 . Recent The roles of mast cells and IgE in regulating Th2 responses in allergic disease in humans are not as clearly established but there is some evidence for such a connection. Testing the immunomodulatory effects of IgE blockade in OIT with food-allergic subjects offers an opportunity to test this question. In humans, omalizumab has been reported to facilitate more aggressive up-dosing, while also reducing allergic reactions during the course of the treatment –138. Str+ APCs. These include Langerhans cells and inflammatory dendritic cells (which do not contain Birbeck granules) in the epidermis, and dermal dendritic cells. FcϵRI is markedly upregulated on these cell types during allergic flares. However, the lack of FcϵRI expression by murine APCs has made it challenging to investigate whether it, like CD23, might promote T cell responses in vivo. Mice with humanized expression of FcϵRI, using an FcϵRI α−chain transgene driven by the CD11c promoter constitutively active in APCs, have proven useful in answering this question. These animals were used to show that allergen-specific IgE, acting via FcϵRI on APCs, instructed naïve T cells to differentiate into Th2 cells, resulting in augmented allergen-specific Th2 responses in vivo  and CD23-dependent process, activating them for efficient antigen presentation  and the avidity of the pooled IgE for antigen affect the efficiency of facilitated antigen presentation . In this process, heavy chains of IgG4 antibodies can separate into half antibodies, each consisting of one heavy chain and one light chain. Half antibodies originating from different parent IgG4s can combine to form bispecific antibodies . This rePerhaps the most compelling evidence for a role of IgG antibodies in regulating food allergen responsiveness has come from OIT studies. In OIT, an allergenic food is administered orally in daily doses that incrementally increase, often up-dosing weekly, over the course of several months. Upon completion of OIT, patients can typically tolerate significant amounts of the allergen without reaction. As this acquired ability to ingest the food is temporary and Tregs which maintain immunological tolerance at the T cell level have not been implicated, this state is referred to as food “unresponsiveness” rather than tolerance . MaintenA clear clue as to the identity of the suppressive factor has been provided by the highly consistent observation that OIT of foods, including milk, egg, and peanut, induces strong food allergen-specific IgG4 responses –172. The-/- BMMCs, indicating a requirement for this receptor in IgG-mediated inhibition. At high doses of IgG, however, inhibition of mast cell activation can be exerted even in cells lacking FcγRIIb, indicating that, in addition to sending negative signals via FcγRIIb, IgG can also act as a blocking antibody, sterically preventing the interaction of allergen with FcϵRI-bound IgE , which is extruded upon degranulation, is a sensitive marker of mast cell activation. Within minutes of exposure to allergen, mast cells degranulate and an increase in LAMP-1 on the cell surface can be detected. Burton and colleagues reported that sera from mice that underwent OIT for peanut allergy can inhibit the peanut-induced activation of BMMCs sensitized with specific IgE in an IgG-dependent manner . In addi IgE see Figure 1 IgE see . Prior tin vivo, particularly to anaphylaxis, has been demonstrated using mouse models. FcγRIIb-deficient mice have provided an excellent genetic tool to analyze this biology. Sensitized FcγRIIb-deficient mice exhibit enhanced anaphylaxis upon allergen challenge , Burton and colleagues analyzed suppressive activity in the serum of peanut-allergic patients who underwent OIT . In the Similar observations were subsequently reported by Santos et al. in a separate peanut-allergic cohort. Rather than using basophils from non-allergic donors to query the OIT sera, this group used the human mast cell line, LAD2 . They to+ food-tolerant subjects and those who have completed OIT has seeded interest in potential therapeutic applications of IgG in food allergy. Although inhibitory food-specific IgG antibodies are still in development by pharmaceutical companies, and there are not yet any direct data regarding the efficacy of IgG in preventing food anaphylaxis, there are preclinical studies that support the approach. Burton and colleagues recently developed a humanized mouse model in which NOD-scid IL2Rγnull mice , given human CD34+ stem cells, exhibit robust T cell expansion, including Foxp3+CD25+ Tregs, IFNγ+ Th1 cells and IL-4+ Th2 cells, and B cell engraftment  that was associated with atopy and IgE production . FunctioIl4raF709 mice, a strain rendered inherently atopic by knock-in of a variant IL-4R α-chain  and MMCP-1 release. However, the protective effects of IgG were dramatically amplified in mice receiving OVA-specific IgG during their OIT with complete abrogation of anaphylaxis and markedly blunted Th2 and IgE responses  cells are required for antibody isotype switching by B cells , 202. ILFel d 1-specific monoclonal IgG4 antibodies in humans prevented symptoms following cat allergen exposure. The dose of IgG4 results in levels in plasma that are in vast excess of the circulating IgE, comparable to the IgG levels induced during successful immunotherapy. In as little as 8 days following injection of Fel d 1 specific IgG4, subjects challenged with cat allergen had a decrease in total nasal symptom score and an increase in peak inspiratory flow compared to controls. By day 29, their skin prick test reactivity was decreased  have increased susceptibility to sensitization and food allergy , 213. Get manner , 11, 213via FcϵRI and in exerting the immunomodulatory effects of IgG requires further study. We believe that investigations in the field have been skewed by the exclusive availability of IgG4 reagents in the ImmunoCAP platform used by most clinical investigators. This has prevented consideration and analysis of the other isotypes. However, as noted in this review, all food-specific antibodies of all four IgG isotypes are induced during OIT and binding to FcγRIIb is clearly not specific to IgG4, begging the question of whether IgG1-3 contribute.The specific contribution of IgG4 relative to other IgG isotypes in the inhibition of IgE-mediated effector cell activation via a number of ways. These observations have been harnessed to engineer antibodies against specific allergen epitopes, and to design small molecules that can aggregate FcγRIIb to FcϵRI-bound IgE bound to antigen. As our understanding of the roles of IgG antibodies both in preventing IgE-triggered anaphylaxis and in regulating Th2 immune responses continues to evolve and as approaches for engineering both allergen-specific and broad-spectrum therapeutics are further developed, we are optimistic that effective strategies will emerge for prevention and treatment in the worldwide problem of food allergies.As presented in this review, data correlating allergen-specific IgG with protection against food allergy phenotypes and the role of FcγRIIb in blocking mast cell and basophil activation has been repeatedly shown by many groups. Though the antibody and the receptor can work together to block the activity of effector cells, research has also shown that they are also capable of exerting inhibitory effects independent of one another Under the supervision of HCO, the manuscript was written by CK with YSEA and OLL. All authors contributed to the article and approved the submitted version.BioRender.com.This work was supported by NIH grant 1R01AI119918 (HO). CK is supported by a postdoctoral fellowship from the Fonds de recherche du Québec—Santé (258617 and 285834). All figures were created with The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Despite the recognition of food allergies as a significant cause of morbidity and a growing public health burden worldwide, there are no epidemiological studies addressing food sensitization and allergy in Jordan.To conduct an epidemiological study that retrospectively analyzes sensitization data from 3463 patients with suspected immunoglobulin E (IgE)‐mediated food allergic reactions who performed specific IgE tests in our laboratories in Jordan.Specific IgE (s‐IgE) tests were analyzed for patients who performed enzyme allegro‐sorbent testing based on either self‐reported food allergy or upon physician's request.2.3% of the analyzed samples were cross‐reactive to carbohydrate determinants. A quarter of the patients were sensitized to one or more food allergens, with males having higher odds of being s‐IgE positive. Furthermore, a higher prevalence of sensitization was seen in infants and children compared with adults. s‐IgE was most frequently found against cow milk (11.2%), pistachio (4.9%), soybean (4.6%), cherry (4.4%), and orange (4.4%). Interestingly, the s‐IgE class distribution profile of pistachio differed from the rest of the top hits being skewed away from the weak class 1 leaning more towards higher IgE classes. Food allergen sensitization was age group‐dependent: milk, tree nuts, and eggs were the main food groups causing sensitization in infants, while it was fruits followed by milk in children and adults.Our work represents the first epidemiological study addressing food sensitization in Jordan. This study lays a solid foundation for future studies that can help better guide food allergy diagnosis, patient dietary modifications, and food elimination plans, as well as assist decision‐makers in the region to develop national strategies for an efficient and sustainable healthcare system. This is the first epidemiological study to investigate the prevalence of food sensitization in Jordan, and one of very few in the Middle East region. We report food‐specific immunoglobulin E (IgE) sensitization and carbohydrate determinant prevalence to a large number of food items in a sizeable and representative cohort of patients suspected of IgE‐mediated food allergic reactions in Jordan. This study reveals gender and age group‐dependent differences in the prevalence of food IgE sensitization, as well as a unique s‐IgE class distribution profile for pistachio, being skewed towards higher IgE classes. Nevertheless, ingested food may cause adverse immunological responses, especially to protein elements.FA incidence is increasing worldwide.There are no epidemiological studies regarding FS and FA in Jordan, and data on the prevalence of FA in the Middle East is fragmented and limited.In this manuscript, data collected retrospectively over the span of 5 years (August 2014‐August 2019) about the age, gender, and s‐IgE FS of patients in Jordan were analyzed. This study is the first epidemiological study coming from Jordan regarding IgE sensitization to a wide range of allergens belonging to foods consumed in the country.22.1Food allergen s‐IgE levels were determined semi‐quantitatively via in vitro EAST, using the IVD‐approved and CE‐certified EUROLINE immunoblot strip “Food Turkey 1” . All strips were incubated in a semi‐automated fashion using EUROBlotMaster (Euroimmun) according to manufacturer's instructions. Briefly, test strips coated with 34 food allergens , in addition to a cross‐reactive carbohydrate determinant (CCD) and an indicator band were moistened and then incubated with 400 µL of undiluted patient serum to bind s‐IgE antibodies present. Subsequently, the bound s‐IgE antibodies were detected using an enzyme‐linked anti‐human IgE catalysing a color reaction. Strips were scanned and then analyzed for EAST class using EUROLineScan software (Euroimmun). A negative result where no s‐IgE is detected was denoted by class 0, while positive results were denoted by class 1 to 6, with low levels classified as class 1 and extremely high levels as class 6.2.2In accordance with the ethical approval number IRB/7/2019 issued by the Cell Therapy Center (CTC), existing patient‐related data used in this study, namely gender, age, and s‐IgE test results were collected retrospectively from Biolab Diagnostic laboratory's laboratory information management system (LIMS), in such a manner that participants cannot be identified directly or through any identifiers linked to them. Inclusion criteria involved patients of both sexes and all ages with a suggestive history of IgE‐mediated FA, tested for s‐IgE upon their request (69%), or that of their treating physician (31%), during the period from August 2014 to August 2019. Samples were obtained from patients who either physically presented to any of the 18 Biolab Diagnostic Laboratory branches in Jordan , or presented to any non‐Biolab laboratory in Jordan that refers blood samples for s‐IgE testing to Biolab's immunology department . All CCD‐positive patients were excluded from the study (83 CCD‐positive tests out of 3647 tests performed during the indicated study period).2.3χ2 goodness of fit test with Yates' correction). The significance of the difference in IgE positivity between age groups was calculated using a one‐way analysis of variance followed by multiple comparisons. P less than .05 and lower were deemed statistically significant.All graphs and statistical analyses were performed using GraphPad Prism (Version 8.0.2), 2019. Significance of the correlation between gender and IgE positivity was tested using contingency table analyses , while 53.8% were females , to account for any new sensitizations appearing between tests due to possible dietary modifications or pollen sensitization. Results indicated that cow milk was the top hit , followed by cow milk, UHT (5.2%), pistachio (4.9%), soybean (4.6%), cherry (4.4%), and orange (4.4%). Lowest s‐IgE prevalence was seen for gluten (0.3%), fish mix (0.4%), olive (0.4%), cocoa bean (0.5%), and onion (0.8%). s‐IgE prevalence data for the various food allergens in all age groups combined was reported in descending order  and hazelnut in infants and children, peanut in infants and adults, and peach and apple in adults (Table S1).3.3The s‐IgE class distribution for the top three food allergens of all ages combined as well as for the individual age groups was then analyzed Figure . In the Furthermore, to shed light on the potential clinical relevance of our data, the top ten food allergens obtained in Figure 3.4To achieve a broader picture of the FS results obtained, we arranged the food items found in the immunoblot panel into food groups (Table S3). Results of patients from all age groups combined revealed that the food group with the highest sensitization rates was fruits (25.3%) (top two fruits being cherry and orange) followed by milk (16%), while the lowest sensitization rates towards cereals (6.3%) and vegetables . Interestingly, these two cities alone host around two‐thirds of the total Jordanian population, making our patient cohort both relevant and representative.Patients with positive IgE antibodies against CCDs were excluded from the study since these antibodies are frequently nonspecific and often lack clinical relevance.Close to a quarter of our total study population was IgE‐sensitized to one or more food allergens. This finding is very similar to a report from Lebanon where 21% of patients with suspected IgE‐mediated food allergic reactions were positive for s‐IgE when tested upon their physician's request.As far as FA is concerned, an imbalance in the direction of infant and childrenThe most commonly encountered s‐IgE antibodies for all age groups combined were against cow milk (including UTH processing), pistachio, and soybean. Interestingly, cow milk and pistachio were seen among the top three hits in infants and children but not adults, while soybean was consistently observed in the top 10 list of all three age categories .Several recent studies shed light on the significance of sesame seed allergy in the Middle East,Very few studies explored the most commonly used tools in the diagnosis of FA in the Middle East. A Lebanese study published in 2015 found out that medical professionals mainly ordered s‐IgE blood tests (55%), followed by food elimination based on clinical history (25%), followed by skin prick testing (20%).This heavy dependence on s‐IgE testing in FA diagnosis calls for the design of customized panels that account for the particularities of the society in question. In this study, we used the EAST panel customized for nearby Turkey because all the food items it featured were popular in the Jordanian diet (with the exception of the fish mix items). This panel, however, understandably lacks some food items highly consumed in Jordan such as chickpeas, pine nuts, thyme, and certain spices. To address this, our group is currently working on generating an s‐IgE panel relevant to Jordan, building on the prevalence data obtained in this study and our knowledge of the country's dietary habits.Despite the usefulness of our findings, this study has some limitations. The retrospective nature of this study makes it very difficult to correlate the sensitization data to patients' clinical history. It is also not possible to provide numerical data regarding the clinical manifestations which ultimately led our patients to perform the food s‐IgE tests.Nevertheless, given that it is generally accepted that an s‐IgE class greater than 2 is interpreted as clinically significant,Finally, new studies to assess the FS and FA status in Jordan at the population level are warranted. Skin prick test results will help corroborate our s‐IgE findings, while double‐blind, placebo‐controlled food challenges are much needed to establish the prevalence of FA in Jordan. Such studies are crucial in identifying the most common food allergens taking into consideration the cultural and nutritional habits of the Jordanian people. Our study represents an important starting point for future work that can facilitate the diagnosis of food allergies in Jordan and the Middle East, and better guide patients' dietary changes and food elimination plans. This study is a significant step forward towards a better understanding of the nature and magnitude of FS in Jordan. Future studies stemming from it will undoubtedly aid in the better assessment of FA in Jordan and can help the Jordanian health authorities make informed decisions as they formulate long‐term strategic health plans concerning FA in the country. We also hope this study and similar ones will prompt the decision‐makers in Jordan to make epinephrine autoinjectors commercially available to all those whose life might depend on it.Ethical approval  to conduct the study was obtained from the clinical research office of the Cell Therapy Center, University of Jordan.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.


Here, we carried this idea forward and introduced the formation of ice PSCs and related dehydration into the sedimentation module of CLaMS. Both processes change the simulated chemical composition of the lower stratosphere. Due to the Lagrangian transport scheme, NAT and ice particles move freely in three-dimensional space. Heterogeneous NAT and ice nucleation on foreign nuclei as well as homogeneous ice nucleation and NAT nucleation on preexisting ice particles are now implemented into CLaMS and cover major PSC formation pathways.Polar stratospheric clouds (PSCs) and cold stratospheric aerosols drive heterogeneous chemistry and play a major role in polar ozone depletion. The Chemical Lagrangian Model of the Stratosphere (CLaMS) simulates the nucleation, growth, sedimentation, and evaporation of PSC particles along individual trajectories. Particles consisting of nitric acid trihydrate (NAT), which contain a substantial fraction of the stratospheric nitric acid  is in very good agreement with MLS observations. In summary, a conclusive agreement between CLaMS simulations and a variety of independent measurements is presented.We show results from the Arctic winter 2009/2010 and from the Antarctic winter 2011 to demonstrate the performance of the model over two entire PSC seasons. For both hemispheres, we present CLaMS results in comparison to measurements from the Cloud-Aerosol Lidar with Orthogonal Polarization (CALIOP), the Michelson Interferometer for Passive Atmospheric Sounding (MIPAS), and the Microwave Limb Sounder (MLS). Observations and simulations are presented on season-long and vortex-wide scales as well as for single PSC events. The simulations reproduce well both the timing and the extent of PSC occurrence inside the entire vortex. Divided into specific PSC classes, CLaMS results show predominantly good agreement with CALIOP and MIPAS observations, even for specific days and single satellite orbits. CLaMS and CALIOP agree that NAT mixtures are the first type of PSC to be present in both winters. NAT PSC areal coverages over the entire season agree satisfactorily. However, cloud-free areas, next to or surrounded by PSCs in the CALIOP data, are often populated with NAT particles in the CLaMS simulations. Looking at the temporal and vortex-averaged evolution of HNO In the lower stratosphere, PSCs provide surfaces for heterogeneous reactions activating chlorine reservoir species and thus accelerating ozone loss . Even thThanks to the Montreal Protocol and its amendments and adjustments, concentrations of ozone-depleting substances are now decreasing continuously , 2018 anPSCs are supposed to consist of liquid supercooled ternary solution (STS) droplets, solid nitric acid trihydrate (NAT) particles, and/or solid ice particles . Their fHere, we present new developments extending the sedimentation module of the Chemical Lagrangian Model of the Stratosphere (CLaMS). We added ice PSC particles to complete the Lagrangian PSC scheme, which allows comparisons to PSC measurements and simulations of de- and rehydration in the Arctic and Antarctic to be performed. To demonstrate the performance of the new CLaMS ice sedimentation module, we have chosen two distinct winters, one Arctic and one Antarctic winter. The Arctic winter 2009/2010 shows widespread ice PSCs during mid-January and is therefore ideally suited to test our new ice scheme. Additionally, the Arctic winter has been the focus of the intensive RECONCILE aircraft campaign, which took place from January until March 2010 . The 2012ζ). At pressure levels lower than 300 hPa, ζ can be interpreted as potential temperature (θ). Towards higher pressure levels, ζ transforms from an isentropic to a pressure-based coordinate  is a global, three-dimensional chemical transport model (CTM) based on the Lagrangian principle . CLaMS iordinate . Mixing ordinate . Wherevestrength . The senstrength . Additiostrength . The detζ < 380 K and PV < 2) within this study with an ice freezing threshold of 100 %. Within the stratosphere, the new ice parameterization of the sedimentation module takes over.The CLaMS cirrus module is used in the regular CLaMS setup to calculate the dehydration of air masses at the tropopause level. This mechanism can be implemented by either using a temperature-dependent parameterization for heterogeneous ice freezing  or by a 2.1The CLaMS sedimentation module offers the possibility to enhance simulations of the polar winter stratosphere by PSC cloud formation and corresponding particle sedimentation. The module was developed by 3 are calculated following 2O vapor pressures are calculated according to 2O and HNO3 is carried out by taking into account a weighted distance to the three nearest neighbors each above and below , a fixed number of ice particles may nucleate. Ice particle number densities are determined with the help of a look-up table, as done in x axis) and supersaturation (color-coded) defines the number of foreign nuclei initiating ice particle nucleation, which is therefore equal to the number of nucleated ice particles (y axis on the right side of the figure). With decreasing temperature and increasing supersaturation, more contact angles can be activated (y axis on the left side of the figure) and the number density of nucleated ice particles increases. The size of the nucleated ice particles is often determined by equilibrium conditions. While nitric acid uptake by micron-sized particles needs hours, water is in equilibrium on the timescales of seconds  = 10 cm−3.Homogeneous nucleation of ice crystals from supercooled aqueous solution droplets was described by es e.g., . Therefo−3 have been observed (see n(NAT) = 1 cm−3 per nucleation event. NAT particle radii and volume are determined assuming thermodynamical equilibrium. This calculation is a first and easy attempt to include NAT nucleation on preexisting ice particles in a global model and may need refinements in later studies.NAT nucleation on preexisting ice particles is an accepted and often confirmed pathway of NAT formation . Downstr2.2It has been shown in several studies that small-scale temperature fluctuations are ubiquitous in the atmosphere and play an important role in ice cloud formation e.g., . Even ifW is calculated for each day of the year (DOY) and both hemispheres in the following way:For CLaMS, we make use of parameterizations by 33 and H2O. As part of the NASA-ESA A-Train constellation, both instruments closely follow each other along the same track describing a sun-synchronous polar orbit with a global coverage ranging from 82° N to 82° S , and the Michelson Interferometer for Passive Atmospheric Sounding. Since April 2006, CALIOP has been flying on the CALIPSO satellite measuring high-resolution backscatter profiles from which information about PSC composition can be inferred. MLS was launched in July 2004 on the Aura spacecraft and delivers profiles of HNOto 82° S . MIPAS oto 82° S . The sat3.1T < 200 K) in perpendicular backscatter (βperp) or scattering ratio (R) at 532 nm. The background threshold values  are defined as median plus one median absolute deviation. The thresholds are daily values that vary with potential temperature and are included in the CALIOP v2 PSC data files. A CALIOP measurement sample is defined to be a PSC if either βperp or R exceeds the background threshold plus an uncertainty (σ); i.e. βperp > βperp, threshold + σ(βperp) or R > Rthreshold + σ(R). An outlier in βperp is assumed to contain detectable non-spherical particles. The optical space for non-spherical particles is divided into two general regimes. A dynamical boundary (RNAT|ice) separates NAT mixtures from ice. RNAT|ice is computed from estimates of cloud-free MLS HNO3 and H2O vapors to account for effects of dehydration and denitrification. A data point is classified as ice if R > RNAT|ice and R < 50, and it is further classified as wave ice if R > 50. NAT mixtures are defined to be the opposite of the dynamical boundary with values of R < RNAT|ice. Any NAT mixture with R > 2 and βperp > 2 × 10−5 km−1 sr−1 belongs to the subclass named “enhanced NAT mixtures”. Each data point that is not an outlier in βperp, but is an outlier in R, is classified as STS. A visualization of the PSC classification can be found in the lower panels of During the last decade, CALIOP was the basis of various studies on PSCs e.g., . Within T-matrix calculations to compute scattering of light by STS, NAT, and ice particles as a function of wavelength :A comparison of individual PSC clouds simulated within CLaMS and measured by CALIOP requires a conversion of model results into optical parameters. For every CALIOP data point, we compute a size distribution from CLaMS PSC particles within a radius of 50 km around the point of measurement and attribute this to the observation. As in th e.g., . Prolateth e.g., . To fullng scale . We used2 cm−3 for STS droplets  were applied to classify CLaMS model results for the comparison with MIPAS. Please note that vertical sampling differences exist between MIPAS (1.5–3 km) and CLaMS (~ 800 m) and that measured and sampled air volumes do not perfectly match.Within this paper, we compared horizontal distributions of PSC composition classes for both hemispheres on single days at constant levels of potential temperature. MIPAS data were processed as described above. For CLaMS, we calculated trajectories from the model results to map those onto the MIPAS measurement locations. As done for the comparison of PSC areal coverage see , we used3.32O and HNO3) via passive measurement of microwave thermal emission from the limb of the Earth’s atmosphere  and βperp agree well with the CALIOP observations. Areas classified as ice lie predominantly within the Tfrost contour line and areas classified as NAT within the TNAT contour line. The maximum values of R – 1 and βperp are in the same order of magnitude and the vertical and horizontal extent of the observed PSC agrees with the model result. The lower panels of βperp, threshold and Rthreshold and the daily mean for RNAT|ice. The spread in the CALIOP data is caused by measurement noise. Although measurement noise is mimicked and added to the modeled data, the spread in the modeled data is slightly less than for the observed values. Those data points are still more confined and do not fill the whole space of the diagram. Enhanced NAT mixtures represent PSCs heterogeneously nucleated in wave ice PSCs. The CALIOP criteria defining enhanced NAT mixtures are conservative and, therefore, the enhanced NAT mixtures subclass is not all-inclusive . The mixed type is chosen such that the simulated surface areas for both, STS and NAT, need to lie above the threshold. Even though ice formation is highly temperature dependent, the spatial pattern of ice PSC occurrence between MIPAS and CLaMS agrees well . Discrepancies between the model and both satellite datasets appear to be related to the relative proportion of STS and NAT PSCs with CLaMS overestimating NAT occurrences. Tfrost. The spread in the modeled data shown in the lower panels (1/R vs. βperp) is slightly less than for the observed values. However, observations and model results lie close together. Overall, over the entire season, CLaMS simulations somewhat underestimate ice occurrences on several occasions  and HNO3 (renitrified) air. They appear below the depleted regions as seen in both MLS observations and CLaMS. The results highlight the new capability of CLaMS to simulate the vertical redistribution of H2O in good agreement with observations. The cirrus module of CLaMS would irreversibly remove the water in the dehydrated areas. In contrast, the new sedimentation module conserves H2O. The signal of sedimentation with subsequent rehydration below is visible until the end of July in the observations as well as in the simulations. Thereafter, a diabatic descent of the rehydrated layer causes water that had originally sedimented from higher latitudes to accumulate in the tropopause region. Vertical velocities are deduced from ERA-Interim diabatic heating rates. In October and November 2011, ERA-Interim indicates positive ascent rates, causing the behavior seen in the simulations in spring. The temporal evolution of both species agrees reasonably well between MLS and CLaMS. The differences between measurements and simulations are quantified in the right panels (2O match very well. The layer of rehydrated air around 350 K potential temperature is slightly lower than in the observations, meaning that H2O mixing ratios are smaller in the simulation than in the observations. A comparison between MLS and CLaMS HNO3 mixing ratios is acceptable but reveals differences. CLaMS HNO3 gas-phase mixing ratios around 500 K potential temperature are lower than the observations for the whole season. However, from August on, a layer of high HNO3 values below 500 K points to the possibility that the redistribution of HNO3 is not efficient enough in the simulation and needs to extend down to lower altitudes. This might explain the simulation of NAT particles in areas which are almost cloud-free in the observations as seen in 3 might be even lower than observed at that time, HNO3 in the model could still be present in the particle phase and could not be redistributed correctly to lower altitudes.The temporal evolution of gas-phase water vapor and nitric acid as measured by MLS and simulated by CLaMS is presented in t panels . The min5We present CLaMS simulations based on a new Lagrangian ice sedimentation scheme focusing on the 2009/2010 Arctic and 2011 Antarctic winters. Previous CLaMS studies solely simulated NAT PSCs and the corresponding denitrification. Here, we extended the model by adding ice PSC particle nucleation, growth, sedimentation, and evaporation. Heterogeneous and homogeneous ice nucleation is included, as well as NAT nucleation on preexisting ice particles. Heterogeneous ice nucleation rates are based on 3, CLaMS shows an uptake of HNO3 from the gas into the particle phase, which is somewhat too large and happens too early in the NH season. The permanent redistribution of HNO3 in the NH is smaller compared to the observations. Also the Antarctic model run shows too little denitrification at lower altitudes towards the end of the winter compared to the observations. These findings point to shortcomings in the simulation of NAT particle sizes in combination with number densities, namely that NAT particle sedimentation should be more efficient in CLaMS. Further studies should try to find better combinations of NAT number densities and sizes with the potential to denitrify the stratosphere more precisely. Heterogeneous NAT nucleation on foreign nuclei and preexisting ice particles has already been implemented and covers most currently discussed routes to form NAT. However, NAT clouds downstream of mountain waves may act as “mother clouds” and individual NAT particles falling out of these clouds in low number densities can grow to large sizes of up to 10 μm (The agreement between CLaMS simulations and the CALIOP, MIPAS, and MLS observations on different temporal and spatial scales is convincing. CLaMS PSC areal coverage presented for both seasons is in good agreement with MIPAS and CALIOP. Similar comparisons between satellite observations and model simulations have been performed in the past e.g., . Howeverto 10 μm . So far,2O observations. To bring this to even better agreement, the level of dehydration in CLaMS could be slightly larger, to lower the minimum values of H2O in the simulations by about 0.5 ppm. So far, ice nucleation on preexisting NAT particles has not been considered in our CLaMS simulations. This pathway will be included in the next CLaMS PSC study. Recently, Simulated ice PSC coverages, in turn, are almost in agreement with the observations as seen in the comparison between CALIOP, MIPAS, and CLaMS. CLaMS simulations miss wave ice clouds as seen in the beginning of the 2009/2010 Arctic winter, likely because ERA-Interim temperature fields are too coarse to resolve the required low values. The small-scale fluctuations added in this study are ubiquitous and not related to specific gravity wave sources such as mountains. A great leap forward would be a change from ERA-Interim to the higher-resolution ERA5 data. This dataset may resolve mountain waves and, once high supersaturations with respect to ice are reached, a revision of the homogeneous ice nucleation parameterization is meaningful. The modeled extent of the dehydration signal fits with the MLS HDespite deficiencies, the overall agreement between CLaMS and different PSC and trace gas measurements is convincing. The advanced microphysical PSC scheme, which includes now STS, NAT, and ice PSCs, therefore represents a major improvement of the representation of cloud physics in CLaMS. Further studies will benefit from this development and may provide more insights into the occurrence and importance of PSC types and their formation mechanisms.