Synthesis and biological evaluation of lipid A derived from commensal Bacteroides.

The inflammation-inducing properties of lipopolysaccharides (LPS) of Gram-negative bacteria reside in their lipid A moiety. Bacillus fragilis, which is a commensal Gram-negative bacterium, biosynthesises lipid A that is structurally distinct from that of E. coli and other enteric bacteria. It is composed of a ß1,6-linked glucosamine (GlcN) disaccharide that is only phosphorylated at the anomeric center. The major species of B. fragilis has five fatty acids and the amine of the distal GlcN moiety carries the unusual (R)-3-(13-methyltetradecanoyloxy)-1.5-methylhexadecanoic acid. A recent study indicates that the LPS of B. fragilis has anti-viral activity by selective induction of interferon (IFN)-ß and is protective in mouse models of vesicular stomatitis virus (VSV) and influenza A. Heterogeneity in the structures of LPS and lipid A and possible contamination with other inflammatory components make it difficult to unambiguously define the immune-modulatory properties of LPS or lipid A. Therefore, we developed a synthetic approach for the preparation of the unusual major lipid A species derived from B. fragilis, which includes a synthetic approach for (R)-3-(13-methyltetradecanoyloxy)-1.5-methylhexadecanoic acid by the Wittig olefination to install the terminal isopropyl moiety. The proinflammatory and antiviral responses of synthetic B. fragilis lipid A were investigated in several cell lines and primary human monocytes by examining the production of interleukin (IL)-6 and IFN-ß. It was found that B. fragilis does not induce the production of IL-6 and IFN-ß but can partially antagonize the production of pro-inflammatory cytokines induced by E. coli LPS and lipid A.

Why Lungs Keep Time: Circadian Rhythms and Lung Immunity.

Circadian rhythms are daily cycles in biological function that are ubiquitous in nature. Understood as a means for organisms to anticipate daily environmental changes, circadian rhythms are also important for orchestrating complex biological processes such as immunity. Nowhere is this more evident than in the respiratory system, where circadian rhythms in inflammatory lung disease have been appreciated since ancient times. In this focused review we examine how emerging research on circadian rhythms is being applied to the study of fundamental lung biology and respiratory disease. We begin with a general introduction to circadian rhythms and the molecular circadian clock that underpins them. We then focus on emerging data tying circadian clock function to immunologic activities within the respiratory system. We conclude by considering outstanding questions about biological timing in the lung and how a better command of chronobiology could inform our understanding of complex lung diseases.

Hypoxia and HIFs in Ewing sarcoma: new perspectives on a multi-facetted relationship.

Hypoxia develops during the growth of solid tumors and influences tumoral activity in multiple ways. Low oxygen tension is also present in the bone microenvironment where Ewing sarcoma (EwS) - a highly aggressive pediatric cancer - mainly arises. Hypoxia inducible factor 1 subunit alpha (HIF-1-a) is the principal molecular mediator of the hypoxic response in cancer whereas EWSR1::FLI1 constitutes the oncogenic driver of EwS. Interaction of the two proteins has been shown in EwS. Although a growing body of studies investigated hypoxia and HIFs in EwS, their precise role for EwS pathophysiology is not clarified to date. This review summarizes and structures recent findings demonstrating that hypoxia and HIFs play a role in EwS at multiple levels. We propose to view hypoxia and HIFs as independent protagonists in the story of EwS and give a perspective on their potential clinical relevance as prognostic markers and therapeutic targets in EwS treatment.

EAACI Molecular Allergology User's Guide 2.0.

Since the discovery of immunoglobulin E (IgE) as a mediator of allergic diseases in 1967, our knowledge about the immunological mechanisms of IgE-mediated allergies has remarkably increased. In addition to understanding the immune response and clinical symptoms, allergy diagnosis and management depend strongly on the precise identification of the elicitors of the IgE-mediated allergic reaction. In the past four decades, innovations in bioscience and technology have facilitated the identification and production of well-defined, highly pure molecules for component-resolved diagnosis (CRD), allowing a personalized diagnosis and management of the allergic disease for individual patients. The first edition of the "EAACI Molecular Allergology User's Guide" (MAUG) in 2016 rapidly became a key reference for clinicians, scientists, and interested readers with a background in allergology, immunology, biology, and medicine. Nevertheless, the field of molecular allergology is moving fast, and after 6 years, a new EAACI Taskforce was established to provide an updated document. The Molecular Allergology User's Guide 2.0 summarizes state-of-the-art information on allergen molecules, their clinical relevance, and their application in diagnostic algorithms for clinical practice. It is designed for both, clinicians and scientists, guiding health care professionals through the overwhelming list of different allergen molecules available for testing. Further, it provides diagnostic algorithms on the clinical relevance of allergenic molecules and gives an overview of their biology, the basic mechanisms of test formats, and the application of tests to measure allergen exposure.

Distinction between peanut allergy and tolerance by characterization of B cell receptor repertoires.

BACKGROUND: Specific IgE against a peanut 2S albumin (Ara h 2 or 6) is the best predictor of clinically relevant peanut sensitization. However, sIgE levels of peanut allergic and those of peanut sensitized but tolerant patients partly overlap, highlighting the need for improved diagnostics to prevent incorrect diagnosis and consequently unnecessary food restrictions. Thus, we sought to explore differences in V(D)J gene transcripts coding for peanut 2S albumin-specific monoclonal antibodies (mAbs) from allergic and sensitized but tolerant donors. METHODS: 2S albumin-binding B-cells were single-cell sorted from peripheral blood of peanut allergic (n=6) and tolerant (n=6) donors sensitized to Ara h2 and/or 6 (≥ 0.1 kU/l) and non-atopic controls (n=5). h 2 and/or 6 (≥ 0.1 kU/l). Corresponding h heavy and light chain gene transcripts were heterologously expressed as mAbs and tested for specificity to native Ara h2 and 6. HCDR3 sequence motifs were identified by Levenshtein distances and hierarchically clustering. RESULTS: The frequency of 2S albumin-binding B cells was increased in allergic (median: 0.01%) compared to tolerant (median: 0.006%) and non-atopic donors (median: 0.0015%, p = 0.008). The majority of mAbs (74%, 29/39) bound specifically to Ara h 2 and/or 6. Non-specific mAbs (9/10) were mainly derived from non-atopic controls. In allergic donors, 89% of heavy chain gene transcripts consisted of VH3 family genes, compared with only 54% in sensitized but tolerant and 63% of non-atopic donors. Additionally, certain HCDR3 sequence motifs were associated with allergy (n = 4) or tolerance (n = 3) upon hierarchical clustering of their Levenshtein distances. CONCLUSIONS: Peanut allergy is associated with dominant VH3 family gene usage and certain public antibody sequences (HCDR3 motifs).

Detection of specific IgE against linear epitopes from Gal d 1 has additional value in diagnosing hen's egg allergy in adults.

BACKGROUND: Although hen's egg allergy is more prevalent in children, up to 0.6% of adults from different European countries suffer from a persistent or newly onset hen's egg allergy, making accurate diagnosis in adults necessary. However, sensitization to hen's egg extracts, components and linear epitopes is solely studied in children. METHODS: Hen's egg allergic (n = 16) and tolerant (n = 19) adults were selected by sensitization towards recombinant components rGal d 1 and/or 3. Sensitization profiles towards egg white and yolk extract and the native components Gal d 1, 2, 3 and 4 were respectively evaluated with the ImmunoCAP or the EUROLINE system. Characterization of linear epitopes was performed with a peptide microarray containing 15mer peptides representing the entire sequence of mature Gal d 1 and 3. RESULTS: Overall, sIgE titres against hen's egg extracts and single components overlapped largely between allergic and tolerant adults. Although the median sIgE/sIgG4 ratio to Gal d 1 was increased in allergic adults, the range was comparable between both groups. Clinically relevant sensitization to Gal d 1 was confirmed by sIgE-binding to the linear epitopes aa30-41, aa39-50 or aa84-95 in 6/13 allergic adults, mainly suffering from objective symptoms. In comparison, these epitopes were recognized by 1/15 tolerant patient. Only a few linear epitopes were detected for Gal d 3, suggesting a greater importance of conformational epitopes for the recognition of Gal d 3. CONCLUSION AND CLINICAL RELEVANCE: Specific IgE-binding to linear epitopes of Gal d 1 is highly specific in identifying hen's egg allergic adults with objective symptoms.

Can alternative epitope mapping approaches increase the impact of B-cell epitopes in food allergy diagnostics?

In vitro allergy diagnostics are currently based on the detection of specific IgE binding on intact allergens or a mixture thereof. This approach has drawbacks as it may yield false-negative and/or false-positive results. Thus, we reviewed the impact of known B-cell epitopes of food allergens to predict transience or persistence, tolerance or allergy and the severity of an allergic reaction and to examine new epitope mapping strategies meant to improve serum-based allergy diagnostics. Recent epitope mapping approaches have been worthwhile in epitope identification and may increase the specificity of allergy diagnostics by using epitopes predominately recognized by allergic patients in some cases. However, these approaches did not lead to discrimination between clinically relevant and irrelevant epitopes so far, since the polyclonal serum IgE-binding epitope spectrum seems to be too individual, independent of the disease status of the patients. New epitope mapping strategies are necessary to overcome these obstacles. The use of patient-derived monoclonal antibodies instead of patient sera for functional characterization of clinically relevant and irrelevant epitope combinations, distinguished by their ability to induce degranulation, might be a promising approach to gain more insight into the allergic reaction and to improve serum-based allergy diagnostics.

Ara h 7 isoforms share many linear epitopes: Are 3D epitopes crucial to elucidate divergent abilities?

BACKGROUND: The peanut allergens Ara h 2, h 6, and h 7 are potent allergens and can trigger severe reactions. Ara h 7 consists of three isoforms differing in their ability to induce basophil degranulation, whereas the ability of Ara h 7.0201 is comparable to Ara h 2 and 6 as shown in previous literature. OBJECTIVE: To identify linear epitopes of Ara h 7.0101, Ara h 7.0201 and Ara h 7.0301 recognized by IgE and IgG4 from patients sensitized to Ara h 7 and to investigate their potential to elucidate divergent abilities of the Ara h 7 isoforms in inducing basophil activation. METHODS: Linear epitopes recognized by IgE and IgG4 were mapped by peptide microarray analysis containing 15-mer peptides of Ara h 2.0201, 6, 7.0101, 7.0201 and 7.0301 and 39 peanut allergic patients sensitized to Ara h 7 (discovery). For validation, 20-mer peptides containing the minimal epitope and surrounding amino acids were incubated with 25 sensitized patients and 10 controls (validation). RESULTS: Three out of 14 linear epitopes were unique for each isoform (Ara h 7.0101: aa 97-109; Ara h 7.0201: aa 122-133; Ara h 7.0301: aa 65-74) but scarcely recognized by IgE. The main linear IgE epitope (aa 51-57) located in the long flexible loop of all Ara h 7 isoforms was bound by antibodies from 31% of the patients (discovery and validation cohort). Regarding IgG4, 55% of the patients recognized an epitope present on all isoforms (aa 55-65), whereas epitope aa 129-137, only present on Ara h 7.0101/0.0301, was recognized by 38% of the patients. Recognition was highly individual, although 20% of the patients recognized any linear epitope neither by IgE nor by IgG4 despite a low mean z-score of ≥ 1.7. Remarkably, only 50% of the patients recognized one or more epitopes by IgE. CONCLUSION & CLINICAL RELEVANCE: Ara h 7 isoforms share many linear epitopes being easily accessible for antibody binding. Unique epitopes, essential to elucidate divergent potencies, were scarcely recognized, suggesting a crucial involvement of conformational epitopes.

Complement component C3 and C5b-9 deposition on hypoxia reperfused endothelial cells by non-HLA antibodies against RhoGDI2: A player involved in graft failure?

Antibodies against Rho GDP-dissociation inhibitor 2 (RhoGDI2) are associated with inferior graft survival in transplant patients receiving a kidney from deceased donors. Although this suggests that these antibodies contribute to graft injury because of ischemia, it remains unknown whether they are also pathogenically involved in the process of graft loss. To study this, we firstly analyzed the IgG subclass profile of anti-RhoGDI2 antibodies in kidney transplant recipients, and whether antibody titers change over time or because of acute rejection. Next, we investigated the expression of RhoGDI2 on primary kidney and lung endothelial cells (ECs) upon hypoxia reperfusion. In addition, the complement-fixing properties of anti-RhoGDI2 antibodies were studied using imaging flow cytometry. Anti-RhoGDI2 antibodies in patients are mainly IgG1, and titers remained stable and seemed not be changed because of rejection. Antibodies against RhoGDI2, which surface expression seemed to increase upon hypoxia reperfusion, co-localized with C3 on ECs. Binding of human IgG1 monoclonal anti-RhoGDI2 antibodies as well as patient derived antibodies, resulted in complement activation, suggesting that these antibodies are complement fixing. This study suggested a potential pathogenic role of anti-RhoGDI2 antibodies in kidney graft loss. During ischemia reperfusion, the ability of these antibodies to fix complement could be one of the mechanisms resulting in tissue injury.

Co-sensitization between legumes is frequently seen, but variable and not always clinically relevant.

Background: Food allergy to peanut and soybean, both legumes, is highly prevalent. The consumption of other legumes and legume protein isolates, some of which may be considered novel foods, is increasing. This may lead to an increase in sensitization and allergy and may pose a risk for legume-allergic (e.g. peanut and soybean) patients due to cross-reactivity. Objective: This study investigated the frequency of co-sensitization and co-allergy between legumes and the role of different protein families. Methods: Six legume-allergic patient groups were included: peanut (n = 30), soybean (n = 30), lupine (n = 30), green pea (n = 30), lentil (n = 17), bean (n = 9). IgE binding to total extracts, protein fractions (7S/11S globulin, 2S albumin, albumin), and 16 individual proteins from 10 legumes (black lentil, blue lupine, chickpea, faba bean, green lentil, pea, peanut, soybean, white bean, and white lupine) was measured by line blot. Results: Co-sensitization varied from 36.7% to 100%. Mono-sensitization was only found in soybean (16.7%), peanut (10%), and green pea-allergic (3.3%) patients. A high frequency of co-sensitization between the 7S/11S globulin fractions of all 10 legumes and individual 7S and 11S globulins was observed. In peanut and soybean-allergic patients, co-allergies for other legumes were uncommon (≤16,7%), while in green pea, lupine, lentil, and bean-allergic patients co-allergy for peanut (64.7%-77.8%) or soybean (50%-64.7%) was frequently seen. Conclusion: Co-sensitization between legumes was high, but generally not clinically relevant. Co-allergy to other legumes was not often seen in peanut- and soybean allergic patients. The 7S and 11S globulins were likely responsible for the observed co-sensitization.

Systematic multi-omics cell line profiling uncovers principles of Ewing sarcoma fusion oncogene-mediated gene regulation.

Ewing sarcoma (EwS) is characterized by EWSR1-ETS fusion transcription factors converting polymorphic GGAA microsatellites (mSats) into potent neo-enhancers. Although the paucity of additional mutations makes EwS a genuine model to study principles of cooperation between dominant fusion oncogenes and neo-enhancers, this is impeded by the limited number of well-characterized models. Here we present the Ewing Sarcoma Cell Line Atlas (ESCLA), comprising whole-genome, DNA methylation, transcriptome, proteome, and chromatin immunoprecipitation sequencing (ChIP-seq) data of 18 cell lines with inducible EWSR1-ETS knockdown. The ESCLA shows hundreds of EWSR1-ETS-targets, the nature of EWSR1-ETS-preferred GGAA mSats, and putative indirect modes of EWSR1-ETS-mediated gene regulation, converging in the duality of a specific but plastic EwS signature. We identify heterogeneously regulated EWSR1-ETS-targets as potential prognostic EwS biomarkers. Our freely available ESCLA (http://r2platform.com/escla/) is a rich resource for EwS research and highlights the power of comprehensive datasets to unravel principles of heterogeneous gene regulation by chimeric transcription factors.

Comparison of Two Strategies to Generate Antigen-Specific Human Monoclonal Antibodies: Which Method to Choose for Which Purpose?

Human monoclonal antibodies (mAbs) are valuable tools to link genetic information with functional features and to provide a platform for conformational epitope mapping. Additionally, combined data on genetic and functional features provide a valuable mosaic for systems immunology approaches. Strategies to generate human mAbs from peripheral blood have been described and used in several studies including single cell sequencing of antigen-binding B cells and the establishment of antigen-specific monoclonal Epstein-Barr Virus (EBV) immortalized lymphoblastoid cell lines (LCLs). However, direct comparisons of these two strategies are scarce. Hence, we sought to set up these two strategies in our laboratory using peanut 2S albumins (allergens) and the autoantigen anti-Rho guanosine diphosphate dissociation inhibitor 2 (RhoGDI2, alternatively 'ARHGDIB') as antigen targets to directly compare these strategies regarding costs, time expenditure, recovery, throughput and complexity. Regarding single cell sequencing, up to 50% of corresponding V(D)J gene transcripts were successfully amplified of which 54% were successfully cloned into expression vectors used for heterologous expression. Seventy-five percent of heterologously expressed mAbs showed specific binding to peanut 2S albumins resulting in an overall recovery of 20.3%, which may be increased to around 29% by ordering gene sequences commercially for antibody cloning. In comparison, the establishment of monoclonal EBV-LCLs showed a lower overall recovery of around 17.6%. Heterologous expression of a mAb carrying the same variable region as its native counterpart showed comparable concentration-dependent binding abilities. By directly comparing those two strategies, single cell sequencing allows a broad examination of antigen-binding mAbs in a moderate-throughput manner, while the establishment of monoclonal EBV-LCLs is a powerful tool to select a small number of highly reactive mAbs restricted to certain B cell subpopulations. Overall, both strategies, initially set-up for peanut 2S albumins, are suitable to obtain human mAbs and they are easily transferrable to other target antigens as shown for ARHGDIB.

Accurate Prediction of Peanut Allergy in One-Third of Adults Using a Validated Ara h 2 Cutoff.

BACKGROUND: The diagnostic value of peanut components is extensively studied in children, but to a lesser extent in adults with suspected peanut allergy. The use of peanut components in daily practice may reduce the need for double-blind placebo-controlled food challenges (DBPCFCs); however, validation studies are currently lacking. OBJECTIVE: To evaluate the diagnostic value of (combined) peanut components and validate a previously found Ara h 2 cutoff level with 100% positive predictive value (PPV) in adults with suspected peanut allergy. METHODS: Adults who underwent a peanut DBPCFC were included: 84 patients from a previous study (2002-2012) and 70 new patients (2012-2019). Specific IgE (sIgE) to peanut extract, Ara h 1, 2, 3, 6, and 8 was measured using ImmunoCAP. Diagnostic value was assessed with an area under the curve (AUC) analysis. RESULTS: In total, 95 (62%) patients were peanut allergic. sIgE to Ara h 2 and Ara h 6 were the best predictors with an AUC (95% confidence interval) of 0.85 (0.79-0.91) and 0.85 (0.79-0.92), respectively. The Ara h 2 cutoff level with 100% PPV (≥1.75 kUA/L) was validated in the 70 new patients. Thirty percent of all included patients could be classified correctly as peanut allergic using this validated cutoff level. CONCLUSION: sIgE to Ara h 2 and Ara h 6 have equally high discriminative ability. Peanut allergy can be predicted accurately in one-third of adults using a validated cutoff level of sIgE to Ara h 2.

Allergy Modulation by N-3 Long Chain Polyunsaturated Fatty Acids and Fat Soluble Nutrients of the Mediterranean Diet.

The Mediterranean diet, containing valuable nutrients such as n-3 long chain poly-unsaturated fatty acids (LCPUFAs) and other fat-soluble micronutrients, is known for its health promoting and anti-inflammatory effects. Its valuable elements might help in the battle against the rising prevalence of non-communicable diseases (NCD), including the development of allergic diseases and other (chronic) inflammatory diseases. The fat fraction of the Mediterranean diet contains bioactive fatty acids but can also serve as a matrix to dissolve and increase the uptake of fat-soluble vitamins and phytochemicals, such as luteolin, quercetin, resveratrol and lycopene with known immunomodulatory and anti-inflammatory capacities. Especially n-3 LCPUFAs such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) derived from marine oils can target specific receptors or signaling cascades, act as eicosanoid precursors and/or alter membrane fluidity and lipid raft formation, hereby exhibiting anti-inflammatory properties. Beyond n-3 LCPUFAs, fat-soluble vitamins A, D, E, and K1/2 have the potential to affect pro-inflammatory signaling cascades by interacting with receptors or activating/inhibiting signaling proteins or phosphorylation in immune cells (DCs, T-cells, mast cells) involved in allergic sensitization or the elicitation/effector phase of allergic reactions. Moreover, fat-soluble plant-derived phytochemicals can manipulate signaling cascades, mostly by interacting with other receptors or signaling proteins compared to those modified by fat-soluble vitamins, suggesting potential additive or synergistic actions by applying a combination of these nutrients which are all part of the regular Mediterranean diet. Research concerning the effects of phytochemicals such as polyphenols has been hampered due to their poor bio-availability. However, their solubility and uptake are improved by applying them within the dietary fat matrix. Alternatively, they can be prepared for targeted delivery by means of pharmaceutical approaches such as encapsulation within liposomes or even unique nanoparticles. This review illuminates the molecular mechanisms of action and possible immunomodulatory effects of n-3 LCPUFAs and fat-soluble micronutrients from the Mediterranean diet in allergic disease development and allergic inflammation. This will enable us to further appreciate how to make use of the beneficial effects of n-3 LCPUFAs, fat-soluble vitamins and a selection of phytochemicals as active biological components in allergy prevention and/or symptom reduction.

IgE-binding to vicilin-like antimicrobial peptides is associated with systemic reactions to macadamia nut.

BACKGROUND: Macadamia nut can induce fatal allergic reactions and changes in dietary habits will raise their consumption in industrialised countries. Until now diagnosis of macadamia nut allergy by sIgE solely relies on the macadamia nut extract, but single components are lacking. METHODS: Macadamia nut proteins recognised by IgE from 2 macadamia nut extract positive sera were identified by mass spectrometry (vicilin-like antimicrobial peptides: VLAP). Sensitisation to macadamia nut extract and heterologously expressed isoform VLAP-2-3 was evaluated in 82 nut allergic (NA) and 27 tolerant (NT) patients (no tree nut allergy reported) comprehending 10 macadamia nut allergic (MA) and 18 explicitly reported macadamia nut tolerant patients (MT), using line blots. Co-sensitisation to additional VLAP isoforms and other vicilins was evaluated in 8 MA, 12 MT and 14 NA patients sensitised to VLAP-2-3. Functional properties were determined by indirect basophil activation. RESULTS: Even though proteins recognised by IgE were identified as VLAP-2-1, 2-2 and 2-3, only peptides specifically belonging to VLAP-2-3 were detected by mass spectrometry. The macadamia nut extract was recognised by 33% of NA patients (27/82) including 3 MA patients and 26% of NT patients (7/27, 3 MT). Similarly, 29% of NA (24/82) patients showed partly strong sIgE-binding to VLAP-2-3 including 3 MA patients with systemic reactions to macadamia nut. Contrary, VLAP-2-3 was recognised by only 2 NT (1 MT) patients (7%) with very low sIgE titres. Simultaneous recognition of the isoforms VLAP-2-1 and 2-2 was observed in all patients positive for VLAP-2-3 with partly reduced sIgE titres in 59% of these patients. Additionally, all three VLAP isoforms were able to repeatedly induce BAT reactivity upon sensitisation with a MA serum. CONCLUSION: VLAP proteins are the first described macadamia nut components with serological and functional allergenic properties and they are associated with systemic reactions to macadamia nut.

Adventures in spacetime: circadian rhythms and the dynamics of protein catabolism.

Circadian rhythms help cells to organize complex processes, but how they shape the kinetics of protein catabolism is unclear. In a recent paper, we employed proteomics to map daily biological rhythms in autophagic flux in mouse liver, and correlated these rhythms with proteasome activity. We also explored the effect of inflammation caused by endotoxin on autophagy dynamics. Our data provide insight into how circadian rhythms serve as a framework for connecting the spatial, temporal, and metabolic aspects of autophagy at a system-wide level. Our observations also have implications for how to optimize autophagy-directed therapies in patients.

Sesame oleosins are minor allergens.

BACKGROUND: In daily practice, one-third of sesame allergic patients, confirmed by clinical history or food challenge, do not show any detectable specific IgE using current diagnostics. Currently used sesame extracts are water-based and therefore lacking hydrophobic proteins like oleosins. Oleosins, the stabilizer of lipid droplets in plants, are described as allergens in sesame, peanut and hazelnut. In this study, we examine the role of oleosins in sesame allergy and their potential cross-reactivity between sesame and (pea)nuts. METHODS: Specific IgE and IgG sensitisation to native and heterologously expressed sesame components and oleosins from other nuts, free of seed storage proteins, was assessed by line blot and sera from 17 sesame allergic patients without detectable specific IgE sensitisation to sesame, and compared to 18 sesame allergic and 13 tolerant patients with specific IgE sensitisation to sesame. RESULTS: Sesame allergic patients without sensitisation showed no specific IgE to the tested sesame oleosins or components. Low levels of specific IgE to sesame oleosins were detected in 17% of sesame allergic and 15% of tolerant patients with sIgE sensitisation. Oleosins were recognised by serum IgG from multiple patients confirming immune reactivity and excluding technical issues leading to lack of specific IgE-binding to oleosins. CONCLUSION: Sesame oleosins are minor allergens and appear to have no additonal value in diagnosing sesame allergy in adults based on sIgE and sIgG detection. There is a high need for additional diagnostic tools in those patients to minimize the number of required food challenges.