Targeting cow's milk allergy using hypoallergenic protein-polyphenol formulas: A proof of concept.

Cow's milk allergy caused by a hypersensitivity to milk proteins has increased over the last years. Hypoallergenic responses can be induced by altering the structure of such proteins with chemical modifiers; this paper is about the creation of edible matrices based on polyphenols from orange peel. Some compounds were able to significantly lower the IgE binding from ß-lactoglobulin allergic serum in sensitized patients. Such effects could also be observed for casein allergic serum in patients sensitized to both proteins. A certain inter-individual variability is observed, although polyphenols do actually induce salient structural changes. This indicates that molecular modifications aimed at oral treatments against food allergy may or may not correlate with reduced allergenicity, and hence the necessity for serum monitoring through immunological assays. Overall, the results are promising enough to validate the polyphenol-fortified approach. In addition, this study highlights the upgrading of vegetable waste, consistent with a circular economy in food chemistry.

Long-chain fatty acids block allergic reaction against lipid transfer protein Sola l 7 from tomato seeds.

Due to the benefits of tomato as an antioxidant and vitamin source, allergy to this vegetable food is a clinically concerning problem. Sola l 7, a class I lipid transfer protein found in tomato seeds, has been identified as an allergen linked to severe anaphylaxis. However, the role of lipid binding in Sola l 7-induced allergy remains unclear. Here, the three-dimensional structure of recombinant Sola l 7 (rSola l 7) has been elucidated using nuclear magnetic resonance spectroscopy (NMR). Its interaction with free fatty acids has been deeply studied; fluorescence emission spectroscopy revealed that different long-chain fatty acids interact with the protein, affecting the only tyrosine residue present in Sola l 7. On the contrary, no changes in the overall secondary structure were observed after the analysis of the circular dichroism spectra in the presence of fatty acids. Unsaturated oleic and linoleic fatty acids presented higher affinity and promoted more significant changes than saturated or short-chain fatty acids. 1H-15N HSQC NMR spectra allowed to determine the regions of the protein that were modified when rSola l 7 interacts with the fatty acids, suggesting epitope modification after the interaction. For corroboration, IgG and IgE binding to rSola l 7 were assessed in the presence of free fatty acids, revealing that both IgE and IgG binding were significantly lower than in their absence, suggesting a potential protective role of unsaturated fatty acids in tomato allergy.

Walnut Jug r 1 is Responsible for Primary Sensitization among Patients Suffering Walnut-Hazelnut 2S Albumin Cross-Reactivity.

Walnut and hazelnut coallergy is a frequent manifestation in clinical practice whose molecular basis remains unclear. For this purpose, walnut-hazelnut cross-reactivity was evaluated in 20 patients allergic to one or both tree nuts and sensitized to their 2S albumins. Immunoblotting assays showed that 85% of patients recognized Jug r 1, walnut 2S albumin, which was associated with the development of severe symptoms; 50% of them corecognized hazelnut 2S albumin, Cor a 14. Both allergens were isolated using chromatographic techniques. Inhibition ELISAs revealed that Jug r 1 strongly inhibited the binding of Cor a 14-specific IgE, but Cor a 14 only partially inhibited Jug r 1-specific IgE binding. Our results showed that patients sensitized to walnut/hazelnut 2S albumins were not a homogeneous population. There were patients sensitized to specific epitopes of walnut 2S albumins and patients sensitized to cross-reactive epitopes between walnut and hazelnut, with Jug r 1 being the primary sensitizer.

Identification of New Allergens in Macadamia Nut and Cross-Reactivity with Other Tree Nuts in a Spanish Cohort.

The consumption of macadamia nuts has increased due to their cardioprotective and antioxidant properties. However, this rise is consistent with an increase in the cases of macadamia nut allergy, leading to severe reactions. Although two Macadamia integrifolia allergens (Mac i 1 and Mac i 2) have been identified in Australian and Japanese patients, the allergenic sensitization patterns in Western European populations, particularly in Spain, remain unclear. For this purpose, seven patients with macadamia nut allergy were recruited in Spain. Macadamia nut protein extracts were prepared and, together with hazelnut and walnut extracts, were used in Western blot and inhibition assays. IgE-reactive proteins were identified using MALDI-TOF/TOF mass spectrometry (MS). Immunoblotting assays revealed various IgE-binding proteins in macadamia nut extracts. Mass spectrometry identified three new allergens: an oleosin, a pectin acetylesterase, and an aspartyl protease. Cross-reactivity studies showed that hazelnut extract but not walnut extract inhibited macadamia nut oleosin-specific IgE binding. This suggests that oleosin could be used as marker for macadamia-hazelnut cross-reactivity. The results show an allergenic profile in the Spanish cohort different from that previously detected in Australian and Japanese populations. The distinct sensitization profiles observed highlight the potential influence of dietary habits and environmental factors exposure on allergenicity.

Generation of an Ovomucoid-Immune scFv Library for the Development of Novel Immunoassays in Hen's Egg Detection.

Hen's egg allergy is the second most common food allergy among infants and young children. The possible presence of undeclared eggs in foods poses a significant risk to sensitized individuals. Therefore, reliable egg allergen detection methods are needed to ensure compliance with food labeling and improve consumer protection. This work describes for the first time the application of phage display technology for the generation of a recombinant antibody aimed at the specific detection of hen's ovomucoid. First, a single-chain variable fragment (scFv) library was constructed from mRNA isolated from the spleen of a rabbit immunized with ovomucoid. After rounds of biopanning, four binding clones were isolated and characterized. Based on the best ovomucoid-binding candidate SR-G1, an indirect phage enzyme-linked immunosorbent assay (phage-ELISA) was developed, reaching limits of detection and quantitation of 43 and 79 ng/mL of ovomucoid, respectively. The developed ELISA was applied to the analysis of a wide variety of food products, obtaining a good correlation with a commercial egg detection assay used as a reference. Finally, in silico modeling of the antigen-antibody complex revealed that the main interactions most likely occur between the scFv heavy chain and the ovomucoid domain-III, the most immunogenic region of this allergen.

Food Allergens: When Friends Become Foes-Caveats and Opportunities for Oral Immunotherapy Based on Deactivation Methods.

Food allergies represent a serious health concern and, since the 1990s, they have risen gradually in high-income countries. Unfortunately, the problem is complex because genetic, epigenetic, and environmental factors may be collectively involved. Prevention and diagnoses have not yet evolved into efficacious therapies. Identification and control of allergens present in edible substances hold promise for multi-purpose biomedical approaches, including oral immunotherapy. This review highlights recent studies and methods to modify the otherwise innocuous native proteins in most subjects, and how oral treatments targeting immune responses could help cancel out the potential risks in hypersensitive individuals, especially children. We have focused on some physical methods that can easily be conducted, along with chemo-enzymatic modifications of allergens by means of peptides and phytochemicals in particular. The latter, accessible from naturally-occurring substances, provide an added value to hypoallergenic matrices employing vegetal wastes, a point where food chemistry meets sustainable goals as well.

Clinical Characteristics and Sensitization Profile of Patients Allergic to Cow Epithelium.

INTRODUCTION: Cow epithelium allergy (CEA) has been described in workers highly exposed to cattle, such as farmers and veterinarians, being a health problem in this population since it is their main livelihood. This study aimed to characterize the main clinical manifestations and define the sensitization profile of the cow epithelium-allergic population treated in our health area. METHODS: This is a retrospective study including a total of 34 patients with a clinical diagnosis of CEA, confirmed by skin tests, bovine epithelium-specific IgE levels and allergen-specific conjunctival challenge test in some cases. They were distributed by age, sex, profession, clinical symptoms, specific IgE levels to other mammalian epithelia, pollens, mites, and foods. Immunoblotting was performed with extracts from cow dander, cow body fluids (urine and saliva), bull urine, and 17 sera from immunotherapy-untreated CEA patients. RESULTS: The mean age of the patients was 44 years, with a higher incidence in cattle farmers. Rhinoconjunctivitis occurred in 100% of cases, with 35% having monosensitization to cow epithelium. Sera from most patients detected a 20-kDa IgE-binding band in cow dander, cow saliva, cow urine, and bull urine, corresponding to the major allergen Bos d 2 (bovine lipocalin). In 70% of the patients, a 25-kDa band was detected in cow and bull urine extracts, whose identification by mass spectrometry and investigation with protein databases led to the identification of a Bos taurus lipocalin (UniProt protein ID: A0A3Q1LGU7_BOVIN). CONCLUSION: CEA should be considered in patients exposed to cattle and as a cause of occupational disease. The IgE immunodetection revealed sensitization to a protein present in cow and bull urine (odorant-binding protein) not previously described.

Personalized diagnostic approach and indirect quantification of extravasation in human anaphylaxis.

BACKGROUND: Anaphylaxis is the most acute and life-threatening manifestation of allergic disorders. Currently, there is a need to improve its medical management and increase the understanding of its molecular mechanisms. This study aimed to quantify the extravasation underlying human anaphylactic reactions and propose new theragnostic approaches. METHODS: Molecular determinations were performed in paired serum samples obtained during the acute phase and at baseline from patients presenting with hypersensitivity reactions. These were classified according to their severity as Grades 1, 2 and 3, the two latter being considered anaphylaxis. Tryptase levels were measured by ImmunoCAP, and serum protein concentration was quantified by Bradford assay. Human serum albumin (HSA) and haemoglobin beta subunit (HBB) levels were determined by Western blot and polyacrylamide gel electrophoresis, respectively. RESULTS: A total of 150 patients were included in the study. Of them, 112 had experienced anaphylaxis (83 and 29 with Grade 2 and 3 reactions, respectively). Tryptase diagnostic efficiency substantially improved when considering patients' baseline values (33%-54%) instead of the acute value threshold (21%). Serum protein concentration and HSA significantly decreased in anaphylaxis (p < .0001). HSA levels dropped with the severity of the reaction (6% and 15% for Grade 2 and 3 reactions, respectively). Furthermore, HBB levels increased during the acute phase of all hypersensitivity reactions (p < .0001). CONCLUSIONS: For the first time, the extravasation underlying human anaphylaxis has been evaluated based on the severity of the reaction using HSA and protein concentration measurements. Additionally, our findings propose new diagnostic and potential therapeutic approaches for this pathological event.

LTP Allergy Follow-Up Study: Development of Allergy to New Plant Foods 10 Years Later.

INTRODUCTION: Allergy to nonspecific lipid transfer protein (nsLTP) is the main cause of plant-food allergy in Spain. nsLTPs are widely distributed in the plant kingdom and have high cross-reactivity but extremely variable clinical expression. Little is known about the natural evolution of this allergy, which complicates management. The objective of this study was to assess the development of allergy to new plant foods in nsLTP-sensitized patients 10 years after diagnosis. METHODS: One hundred fifty-one patients showing specific IgE to nsLTP determined by ISAC (Thermofisher) were included. After clinical workup (i.e., anamnesis, skin test, and challenge when needed), these patients were divided into two groups: 113 patients allergic to one or more plant food (74.5%) and 38 patients not allergic to any plant food (25.1%). Ten years later, a telephone interview was conducted to check whether patients had developed additional allergic reactions to plant foods. RESULTS: Ten years after diagnosis, 35 of the 113 (31%) plant-food-allergic patients sensitized to nsLTP reported reactions to new, previously tolerated plant foods, mainly Rosaceae/Prunoideae fruits and nuts followed by vegetables, Rosacea/Pomoideae fruits, legumes, and cereals. Five out of 38 (13.2%) patients previously sensitized to nsLTP but without allergy to any plant food had experienced allergic reactions to some plant food: two to Rosaceae/Prunoideae fruits, two to Rosaceae/Prunoideae fruit and nuts, and one to legumes. CONCLUSION: Patients sensitized to nsLTP developed allergic reactions to other plant foods, mainly Rosaceae-Prunoideae fruits and nuts. This was more frequent among plant-food-allergic patients than among those who had never had plant-food allergy.

Proteomic and Biological Analysis of an In Vitro Human Endothelial System in Response to Drug Anaphylaxis.

Anaphylaxis is a life-threatening systemic hypersensitivity reaction. During anaphylaxis, mediator release by effector cells causes endothelial barrier breakdown, increasing vascular permeability and leakage of fluids, which may lead to tissue edema. Although endothelial cells (ECs) are key players in this context, scant attention has been paid to the molecular analysis of the vascular system, and further analyses of this cell type are necessary, especially in humans. The protein expression pattern of human microvascular ECs was analyzed in response to sera from anaphylactic patients (EC-anaphylaxis) and sera from non-allergic subjects (EC-control) after 2 hours of contact. Firstly, a differential quantitative proteomic analysis of the protein extracts was performed by mass spectrometry using an isobaric labeling method. Second, the coordinated behavior of the identified proteins was analyzed using systems biology analysis (SBA). The proteome of the EC-anaphylaxis system showed 7,707 proteins, of which 1,069 were found to be significantly altered between the EC-control and EC-anaphylaxis groups (p-value < 0.05). Among them, a subproteome of 47 proteins presented a high rate of change (|ΔZq| ≥ 3). This panel offers an endothelial snapshot of the anaphylactic reaction. Those proteins with the highest individual changes in abundance were hemoglobin subunits and structural support proteins. The interacting network analysis of this altered subproteome revealed that the coagulation and complement systems are the main biological processes altered in the EC-anaphylactic system. The comprehensive SBA resulted in 5,512 functional subcategories (biological processes), 57 of which were significantly altered between EC-control and EC-anaphylaxis. The complement system, once again, was observed as the main process altered in the EC system created with serum from anaphylactic patients. Findings of the current study further our understanding of the underlying pathophysiological mechanisms operating in anaphylactic reactions. New target proteins and relevant signaling pathways operating in the in vitro endothelial-serum system have been identified. Interestingly, our results offer a protein overview of the micro-EC-anaphylaxis environment. The relevance of the coagulation, fibrinolytic, contact and complement systems in human anaphylaxis is described. Additionally, the untargeted high-throughput analysis used here is a novel approach that reveals new pathways in the study of the endothelial niche in anaphylaxis.

Characterization of Relevant Biomarkers for the Diagnosis of Food Allergies: An Overview of the 2S Albumin Family.

2S albumins are relevant and often major allergens from several tree nuts and seeds, affecting mainly children and young people. The present study aims to assess how the structural features of 2S albumins could affect their immunogenic capacity, which is essential to comprehend the role of these proteins in food allergy. For this purpose, twelve 2S albumins were isolated from their respective extracts by chromatographic methods and identified by MALDI-TOF mass-spectrometry. Their molecular and structural characterization was conducted by electrophoretic, spectroscopic and in silico methods, showing that these are small proteins that comprise a wide range of isoelectric points, displaying a general high structure stability to thermal treatment. Despite low amino acid sequence identity, these proteins share structural features, pointing conformational epitopes to explain cross-reactivity between them. Immunoblotting with allergic patients' sera revealed those possible correlations between evolutionarily distant 2S albumins from different sources. The availability of a well-characterized panel of 2S albumins from plant-derived sources allowed establishing correlations between their structural features and their allergenic potential, including their role in cross-reactivity processes.

Delineation of the Olive Pollen Proteome and Its Allergenome Unmasks Cyclophilin as a Relevant Cross-Reactive Allergen.

Olive pollen is a major allergenic source worldwide due to its extensive cultivation. We have combined available genomics data with a comprehensive proteomics approach to get the annotated olive tree (Olea europaea L.) pollen proteome and define its complex allergenome. A total of 1907 proteins were identified by LC-MS/MS using predicted protein sequences from its genome. Most proteins (60%) were predicted to possess catalytic activity and be involved in metabolic processes. In total, 203 proteins belonging to 47 allergen families were found in olive pollen. A peptidyl-prolyl cis-trans isomerase, cyclophilin, produced in Escherichia coli, was found as a new olive pollen allergen (Ole e 15). Most Ole e 15-sensitized patients were children (63%) and showed strong IgE recognition to the allergen. Ole e 15 shared high sequence identity with other plant, animal, and fungal cyclophilins and presented high IgE cross-reactivity with pollen, plant food, and animal extracts.