Non-Specific Lipid Transfer Protein Amb a 6 Is a Source-Specific Important Allergenic Molecule in Ragweed Pollen.

Pollen from common ragweed is an important allergen source worldwide and especially in western and southern Romania. More than 100 million patients suffer from symptoms of respiratory allergy (e.g., rhinitis, asthma) to ragweed pollen. Among the eleven characterized allergens, Amb a 6 is a non-specific lipid transfer protein (nsLTP). nsLTPs are structurally stable proteins in pollen and food from different unrelated plants capable of inducing severe reactions. The goal of this study was to produce Amb a 6 as a recombinant and structurally folded protein (rAmb a 6) and to characterize its physicochemical and immunological features. rAmb a 6 was expressed in Spodoptera frugiperda Sf9 cells as a secreted protein and characterized by mass spectrometry and circular dichroism (CD) spectroscopy regarding molecular mass and fold, respectively. The IgE-binding frequency towards the purified protein was evaluated using sera from 150 clinically well-characterized ragweed-allergic patients. The allergenic activities of rAmb a 6 and the nsLTP from the weed Parietaria judaica (Par j 2) were evaluated in basophil activation assays. rAmb a 6-specific IgE reactivity was associated with clinical features. Pure rAmb a 6 was obtained by insect cell expression. Its deduced molecular weight corresponded to that determined by mass spectrometry (i.e., 10,963 Da). rAmb a 6 formed oligomers as determined by SDS-PAGE under non-reducing conditions. According to multiple sequence comparisons, Amb a 6 was a distinct nsLTP with less than 40% sequence identity to currently known plant nsLTP allergens, except for nsLTP from Helianthus (i.e., 52%). rAmb a 6 is an important ragweed allergen recognized by 30% of ragweed pollen allergic patients. For certain patients, rAmb a 6-specific IgE levels were higher than those specific for the major ragweed allergen Amb a 1 and analysis also showed a higher allergenic activity in the basophil activation test. rAmb a 6-positive patients suffered mainly from respiratory symptoms. The assumption that Amb a 6 is a source-specific ragweed allergen is supported by the finding that none of the patients showing rAmb a 6-induced basophil activation reacted with Par j 2 and only one rAmb a 6-sensitized patient had a history of plant food allergy. Immunization of rabbits with rAmb a 6 induced IgG antibodies which strongly inhibited IgE binding to rAmb a 6. Our results demonstrate that Amb a 6 is an important source-specific ragweed pollen allergen that should be considered for diagnosis and allergen-specific immunotherapy of ragweed pollen allergy.

A Quantity-Dependent Nonlinear Model of Sodium Cromoglycate Suppression on Beta-Conglycinin Transport.

Understanding the transport mechanism is crucial for developing inhibitors that block allergen absorption and transport and prevent allergic reactions. However, the process of how beta-conglycinin, the primary allergen in soybeans, crosses the intestinal mucosal barrier remains unclear. The present study indicated that the transport of beta-conglycinin hydrolysates by IPEC-J2 monolayers occurred in a time- and quantity-dependent manner. The beta-conglycinin hydrolysates were absorbed into the cytoplasm of IPEC-J2 monolayers, while none were detected in the intercellular spaces. Furthermore, inhibitors such as methyl-beta-cyclodextrin (MßCD) and chlorpromazine (CPZ) significantly suppressed the absorption and transport of beta-conglycinin hydrolysates. Of particular interest, sodium cromoglycate (SCG) exhibited a quantity-dependent nonlinear suppression model on the absorption and transport of beta-conglycinin hydrolysates. In conclusion, beta-conglycinin crossed the IPEC-J2 monolayers through a transcellular pathway, involving both clathrin-mediated and caveolae-dependent endocytosis mechanisms. SCG suppressed the absorption and transport of beta-conglycinin hydrolysates by the IPEC-J2 monolayers by a quantity-dependent nonlinear model via clathrin-mediated and caveolae-dependent endocytosis. These findings provide promising targets for both the prevention and treatment of soybean allergies.

Utilizing the Banana S-Adenosyl-L-Homocysteine Hydrolase Allergen to Identify Cross-Reactive IgE in Ryegrass-, Latex-, and Kiwifruit-Allergic Individuals.

Food allergies mediated by specific IgE (sIgE) have a significant socioeconomic impact on society. Evaluating the IgE cross-reactivity between allergens from different allergen sources can enable the better management of these potentially life-threatening adverse reactions to food proteins and enhance food safety. A novel banana fruit allergen, S-adenosyl-L-homocysteine hydrolase (SAHH), has been recently identified and its recombinant homolog was heterologously overproduced in E. coli. In this study, we performed a search in the NCBI (National Center for Biotechnology Information) for SAHH homologs in ryegrass, latex, and kiwifruit, all of which are commonly associated with pollen-latex-fruit syndrome. In addition, Western immunoblot analysis was utilized to identify the cross-reactive IgE to banana SAHH in the sera of patients with a latex allergy, kiwifruit allergy, and ryegrass allergy. ClustalOmega analysis showed more than 92% amino acid sequence identity among the banana SAHH homologs in ryegrass, latex, and kiwifruit. In addition to five B-cell epitopes, in silico analysis predicted eleven T-cell epitopes in banana SAHH, seventeen in kiwifruit SAHH, twelve in ryegrass SAHH, and eight in latex SAHH, which were related to the seven-allele HLA reference set (HLA-DRB1*03:01, HLA-DRB1*07:01, HLA-DRB1*15:01, HLA-DRB3*01:01, HLA-DRB3*02:02, HLA-DRB4*01:01, HLA-DRB5*01:01). Four T-cell epitopes were identical in banana and kiwifruit SAHH (positions 328, 278, 142, 341), as well as banana and ryegrass SAHH (positions 278, 142, 96, and 341). All four SAHHs shared two T-cell epitopes (positions 278 and 341). In line with the high amino acid sequence identity and B-cell epitope homology among the analyzed proteins, the cross-reactive IgE to banana SAHH was detected in three of three latex-allergic patients, five of six ryegrass-allergic patients, and two of three kiwifruit-allergic patients. Although banana SAHH has only been studied in a small group of allergic individuals, it is a novel cross-reactive food allergen that should be considered when testing for pollen-latex-fruit syndrome.

The cytoskeletal protein profilin is an important allergen in saltwort (Salsola kali).

Pollen from Salsola kali, i.e., saltwort, Russian thistle, is a major allergen source in the coastal regions of southern Europe, in Turkey, Central Asia, and Iran. S. kali-allergic patients mainly suffer from hay-fever (i.e., rhinitis and conjunctivitis), asthma, and allergic skin symptoms. The aim of this study was to investigate the importance of individual S. kali allergen molecules. Sal k 1, Sal k 2, Sal k 3, Sal k 4, Sal k 5, and Sal k 6 were expressed in Escherichia coli as recombinant proteins containing a C-terminal hexahistidine tag and purified by nickel affinity chromatography. The purity of the recombinant allergens was analyzed by SDS-PAGE. Their molecular weight was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and their fold and secondary structure were studied by circular dichroism (CD) spectroscopy. Sera from clinically well-characterized S. kali-allergic patients were used for IgE reactivity and basophil activation experiments. S. kali allergen-specific IgE levels and IgE levels specific for the highly IgE cross-reactive profilin and the calcium-binding allergen from timothy grass pollen, Phl p 12 and Phl p 7, respectively, were measured by ImmunoCAP. The allergenic activity of natural S. kali pollen allergens was studied in basophil activation experiments. Recombinant S. kali allergens were folded when studied by CD analysis. The sum of recombinant allergen-specific IgE levels and allergen-extract-specific IgE levels was highly correlated. Sal k 1 and profilin, reactive with IgE from 64% and 49% of patients, respectively, were the most important allergens, whereas the other S. kali allergens were less frequently recognized. Specific IgE levels were highest for profilin. Of note, 37% of patients who were negative for Sal k 1 showed IgE reactivity to Phl p 12, emphasizing the importance of the ubiquitous cytoskeletal actin-binding protein, profilin, for the diagnosis of IgE sensitization in S. kali-allergic patients. rPhl p 12 and rSal k 4 showed equivalent IgE reactivity, and the clinical importance of profilin was underlined by the fact that profilin-monosensitized patients suffered from symptoms of respiratory allergy to saltwort. Accordingly, profilin should be included in the panel of allergen molecules for diagnosis and in molecular allergy vaccines for the treatment and prevention of S. kali allergy.

IgE-Mediated Sensitization to Blo t 21 and Blo t 5 Is Associated With Asthma in the Tropics: A Case-Control Study.

BACKGROUND AND OBJECTIVE: Sensitization to Blomia tropicalis is associated with asthma in various tropical and subtropical countries; however, information about the specific molecular components associated with this disease is scarce. Using molecular diagnosis, we sought to identify B tropicalis allergens associated with asthma in Colombia. METHODS: Specific IgE (sIgE) to 8 B tropicalis recombinant allergens (Blo t 2, 5, 7, 8, 10, 12, 13, and 21) was determined using an in-house ELISA system in asthma patients (n=272) and controls (n=298) recruited in a national prevalence study performed in several Colombian cities (Barranquilla, Bogotá, Medellín, Cali, and San Andrés). The study sample included children and adults (mean [SD] age, 28 [17] years). Cross-reactivity between Blo t 5 and Blo t 21 was evaluated using ELISA-inhibition. RESULTS: Specific IgE (sIgE) to 8 B tropicalis recombinant allergens (Blo t 2, 5, 7, 8, 10, 12, 13, and 21) was determined using an in-house ELISA system in asthma patients (n=272) and controls (n=298) recruited in a national prevalence study performed in several Colombian cities (Barranquilla, Bogotá, Medellín, Cali, and San Andrés). The study sample included children and adults (mean [SD] age, 28 [17] years). Cross-reactivity between Blo t 5 and Blo t 21 was evaluated using ELISA-inhibition. CONCLUSION: Although Blo t 5 and Blo t 21 are considered common sensitizers, this is the first report of their association with asthma. Both components should be included in molecular panels for diagnosis of allergy in the tropics.

Cross-Reactivity of Ragweed Pollen Calcium-Binding Proteins and IgE Sensitization in a Ragweed-Allergic Population from Western Romania.

Ragweed pollen allergy is the most common seasonal allergy in western Romania. Prolonged exposure to ragweed pollen may induce sensitization to pan-allergens such as calcium-binding proteins (polcalcins) and progression to more severe symptoms. We aimed to detect IgE sensitization to recombinant Amb a 9 and Amb a 10 in a Romanian population, to assess their potential clinical relevance and cross-reactivity, as well as to investigate the relation with clinical symptoms. rAmb a 9 and rAmb a 10 produced in Escherichia coli were used to detect specific IgE in sera from 87 clinically characterized ragweed-allergic patients in ELISA, for basophil activation experiments and rabbit immunization. Rabbit rAmb a 9- and rAmb a 10-specific sera were used to detect possible cross-reactivity with rArt v 5 and reactivity towards ragweed and mugwort pollen extracts. The results showed an IgE reactivity of 25% to rAmb a 9 and 35% to rAmb a 10. rAmb a 10 induced basophil degranulation in three out of four patients tested. Moreover, polcalcin-negative patients reported significantly more skin symptoms, whereas polcalcin-positive patients tended to report more respiratory symptoms. Furthermore, both rabbit antisera showed low reactivity towards extracts and showed high reactivity to rArt v 5, suggesting strong cross-reactivity. Our study indicated that recombinant ragweed polcalcins might be considered for molecular diagnosis.

Insect Cell-Expressed Major Ragweed Allergen Amb a 1.01 Exhibits Similar Allergenic Properties to Its Natural Counterpart from Common Ragweed Pollen.

Common ragweed pollen allergy has become a health burden worldwide. One of the major allergens in ragweed allergy is Amb a 1, which is responsible for over 90% of the IgE response in ragweed-allergic patients. The major allergen isoform Amb a 1.01 is the most allergenic isoform in ragweed pollen. So far, no recombinant Amb a 1.01 with similar allergenic properties to its natural counterpart (nAmb a 1.01) has been produced. Hence, this study aimed to produce a recombinant Amb a 1.01 with similar properties to the natural isoform for improved ragweed allergy management. Amb a 1.01 was expressed in insect cells using a codon-optimized DNA construct with a removable N-terminal His-Tag (rAmb a 1.01). The recombinant protein was purified by affinity chromatography and physicochemically characterized. The rAmb a 1.01 was compared to nAmb a 1.01 in terms of the IgE binding (enzyme-linked immunosorbent assay (ELISA), immunoblot) and allergenic activity (mediator release assay) in well-characterized ragweed-allergic patients. The rAmb a 1.01 exhibited similar IgE reactivity to nAmb a 1.01 in different IgE-binding assays (i.e., IgE immunoblot, ELISA, quantitative ImmunoCAP inhibition measurements). Furthermore, the rAmb a 1.01 showed comparable dose-dependent allergenic activity to nAmb a 1.01 regarding basophil activation. Overall, the results showed the successful expression of an rAmb a 1.01 with comparable characteristics to the corresponding natural isoform. Our findings provide the basis for an improvement in ragweed allergy research, diagnosis, and immunotherapy.

Insight into the interaction and binding mechanism of a natural nonnutritive sweetener mogroside V with soybean protein isolates based on multi-spectroscopic techniques and computational simulations.

As a natural low-calorie sweetener, Mogroside V (Mog-V) has gradually become one of the alternatives to sucrose with superior health attributes. However, Mog-V will bring unpleasant aftertastes when exceeding a threshold concentration. To investigate the possibility of soy protein isolates (SPIs), namely ß-conglycinin (7S), and glycinin (11S) as flavor-improving agents of Mog-V, the binding mechanism between Mog-V and SPIs was explored through multi-spectroscopy, particle size, zeta potential, and computational simulation. The results of the multi-spectroscopic experiments indicated that Mog-V enhanced the fluorescence of 7S/11S protein in a static mode. The binding affinity of 7S-Mog-V was greater compared with 11S-Mog-V. Particle size and zeta potential analysis revealed that the interaction could promote aggregation of 7S/11S protein with different stability. Furthermore, computational simulations further confirmed that Mog-V could interact with the 7S/11S protein in different ways. This research provides a theoretical foundation for the development and application of SPI to improve the flavor of Mog-V, opening a new avenue for further expanding the market demand for Mog-V.

Trimeric Bet v 1-specific nanobodies cause strong suppression of IgE binding.

Background: Around 20% of the population in Northern and Central Europe is affected by birch pollen allergy, with the major birch pollen allergen Bet v 1 as the main elicitor of allergic reactions. Together with its cross-reactive allergens from related trees and foods, Bet v 1 causes an impaired quality of life. Hence, new treatment strategies were elaborated, demonstrating the effectiveness of blocking IgG antibodies on Bet v 1-induced IgE-mediated reactions. A recent study provided evidence for the first time that Bet v 1-specific nanobodies reduce patients´ IgE binding to Bet v 1. In order to increase the potential to outcompete IgE recognition of Bet v 1 and to foster cross-reactivity and cross-protection, we developed Bet v 1-specific nanobody trimers and evaluated their capacity to suppress polyclonal IgE binding to corresponding allergens and allergen-induced basophil degranulation. Methods: Nanobody trimers were engineered by adding isoleucine zippers, thus enabling trimeric formation. Trimers were analyzed for their cross-reactivity, binding kinetics to Bet v 1, and related allergens, and patients' IgE inhibition potential. Finally, their efficacy to prevent basophil degranulation was investigated. Results: Trimers showed enhanced recognition of cross-reactive allergens and increased efficiency to reduce IgE-allergen binding compared to nanobody monomers. Furthermore, trimers displayed slow dissociation rates from allergens and suppressed allergen-induced mediator release. Conclusion: We generated high-affine nanobody trimers that target Bet v 1 and related allergens. Trimers blocked IgE-allergen interaction by competing with IgE for allergen binding. They inhibited IgE-mediated release of biological mediators, demonstrating a promising potential to prevent allergic reactions caused by Bet v 1 and relatives.

Structural and Immunological Features of PR-10 Allergens: Focusing on the Major Alder Pollen Allergen Aln g 1.

Today, allergies have become a serious problem. PR-10 proteins are clinically relevant allergens that have the ability to bind hydrophobic ligands, which can significantly increase their allergenicity potential. It has been recently shown that not only the birch pollen allergen Bet v 1 but also the alder pollen allergen Aln g 1, might act as a true sensitizer of the immune system. The current investigation is aimed at the further study of the allergenic and structural features of Aln g 1. By using qPCR, we showed that Aln g 1 was able to upregulate alarmins in epithelial cells, playing an important role in sensitization. With the use of CD-spectroscopy and ELISA assays with the sera of allergic patients, we demonstrated that Aln g 1 did not completely restore its structure after thermal denaturation, which led to a decrease in its IgE-binding capacity. Using site-directed mutagenesis, we revealed that the replacement of two residues (Asp27 and Leu30) in the structure of Aln g 1 led to a decrease in its ability to bind to both IgE from sera of allergic patients and lipid ligands. The obtained data open a prospect for the development of hypoallergenic variants of the major alder allergen Aln g 1 for allergen-specific immunotherapy.

Effects of Chlorogenic Acid on Lowering IgE-Binding Capacity of Soybean 7S: Comparison between Covalent and Noncovalent Interaction.

Allergenicity of soybean 7S protein (7S) troubles many people around the world. However, many processing methods for lowering allergenicity is invalid. Interaction of 7S with phenolic acids, such as chlorogenic acid (CHA), to structurally modify 7S may lower the allergenicity. Hence, the effects of covalent (C-I, periodate oxidation method) and noncovalent interactions (NC-I) of 7S with CHA in different concentrations (0.3, 0.5, and 1.0 mM) on lowering 7S allergenicity were investigated in this study. The results demonstrated that C-I led to higher binding efficiency (C-0.3:28.51 ± 2.13%) than NC-I (N-0.3:22.66 ± 1.75%). The C-I decreased the α-helix content (C-1:21.06%), while the NC-I increased the random coil content (N-1:24.39%). The covalent 7S-CHA complexes of different concentrations had lower IgE binding capacity (C-0.3:37.38 ± 0.61; C-0.5:34.89 ± 0.80; C-1:35.69 ± 0.61%) compared with that of natural 7S (100%), while the noncovalent 7S-CHA complexes showed concentration-dependent inhibition of IgE binding capacity (N-0.3:57.89 ± 1.23; N-0.5:46.91 ± 1.57; N-1:40.79 ± 0.22%). Both interactions produced binding to known linear epitopes. This study provides the theoretical basis for the CHA application in soybean products to lower soybean allergenicity.

miRNA Expression Analysis of IPEC-J2 Cells Damaged by Soybean 7S Globulin Reveals ssc-miR-221-5p as the Factor Alleviating Cell Damage.

The most significant and sensitive antigen protein that causes diarrhea in weaned pigs is soybean 7S globulin. Therefore, identifying the primary target for minimizing intestinal damage brought on by soybean 7S globulin is crucial. MicroRNA (miRNA) is closely related to intestinal epithelium's homeostasis and integrity. However, the change of miRNAs' expression and the function of miRNAs in Soybean 7S globulin injured-IPEC-J2 cells are still unclear. In this study, the miRNAs' expression profile in soybean 7S globulin-treated IPEC-J2 cells was investigated. Fifteen miRNAs were expressed differently. The differentially expressed miRNA target genes are mainly concentrated in signal release, cell connectivity, transcriptional inhibition, and Hedgehog signaling pathway. Notably, we noticed that the most significantly decreased miRNA was ssc-miR-221-5p after soybean 7S globulin treatment. Therefore, we conducted a preliminary study on the mechanisms of ssc-miR-221-5p in soybean 7S globulin-injured IPEC-J2 cells. Our research indicated that ssc-miR-221-5p may inhibit ROS production to alleviate soybean 7S globulin-induced apoptosis and inflammation in IPEC-J2 cells, thus protecting the cellular mechanical barrier, increasing cell proliferation, and improving cell viability. This study provides a theoretical basis for the prevention and control of diarrhea of weaned piglets.

Mustard seed major allergen Sin a1 activates intestinal epithelial cells and also dendritic cells that drive type 2 immune responses.

Mustard seeds belong to the food category of mandatory labelling due to the severe reactions they can trigger in allergic patients. However, the mechanisms underlying allergic sensitization to mustard seeds are poorly understood. The aim of this work is to study type 2 immune activation induced by the mustard seed major allergen Sin a1 via the intestinal mucosa, employing an in vitro model mimicking allergen exposure via the intestinal epithelial cells (IECs). Sin a1 was isolated from the total protein extract and exposed to IEC, monocyte derived dendritic cells (DCs) or IEC/DC co-cultures. A system of consecutive co-cultures was employed to study the generic capacity of Sin a1 to induce type 2 activation leading to sensitization: IEC/DC, DC/T-cell, T/B-cell and stem cell derived mast cells (MCs) derived from healthy donors. Immune profiles were determined by ELISA and flow cytometry. Sin a1 activated IEC and induced type-2 cytokine secretion in IEC/DC co-culture or DC alone (IL-15, IL-25 and TSLP), and primed DC induced type 2 T-cell skewing. IgG secretion in the T-cell/B-cell phase was enhanced in the presence of Sin a1 in the first stages of the co-culture. Anti-IgE did not induce degranulation but promoted IL-13 and IL-4 release by MC primed with the supernatant from B-cells co-cultured with Sin a1-IEC/DC or -DC primed T-cells. Sin a1 enhanced the release of type-2 inflammatory mediators by epithelial and dendritic cells; the latter instructed generic type-2 responses in T-cells that resulted in B-cell activation, and finally MC activation upon anti-IgE exposure. This indicates that via activation of IEC and/or DC, mustard seed allergen Sin a1 is capable of driving type 2 immunity which may lead to allergic sensitization.

Group 2 innate lymphoid cells are key in lipid transfer protein allergy pathogenesis.

Background: Immunopathology in food allergy is characterized by an uncontrolled type 2 immune response and specific-IgE production. Recent studies have determined that group 2 innate lymphoid cells (ILC2) participate in the food allergy pathogenic mechanism and their severity. Our objective was to investigate the role of ILC2 in peach-allergic patients due to non-specific lipid transfer protein (Pru p 3) sensitization. Methods: The immune response in peripheral blood mononuclear cells was characterized in lipid transfer protein-allergic patients and healthy controls. We have analyzed the Pru p 3 uptake on ILC2, the expression of costimulatory molecules, and their involvement on the T-cell proliferative response and cytokine production under different experimental conditions: cytokines involved in group 2 innate lymphoid cell activation (IL-33 and IL-25), Pru p 3 as main food allergen, and the combination of both components (IL-33/IL-25+Pru p 3) using cell sorting, EliSpot, flow cytometry, and confocal microscopy. Results: Our results show that Pru p 3 allergen is taken up by group 2 innate lymphoid cells, regulating their costimulatory molecule expression (CD83 and HLA-DR) depending on the presence of Pru p 3 and its combination with IL-33/IL-25. The Pru p 3-stimulated ILC2 induced specific GATA3+Th2 proliferation and cytokine (IL-4, IL-5, and IL-13) production in lipid transfer protein-allergic patients in a cell contact-dependent manner with no changes in Tbet+Th1- and FOXP3+Treg cell differentiation. Conclusions: The results indicate that in lipid transfer protein-allergic patients, the responsible allergen, Pru p 3, interacts with group 2 innate lymphoid cells, promoting a Th2 cell response. Our results might be of interest in vivo, as they show a role of group 2 innate lymphoid cells as antigen-presenting cells, contributing to the development of food allergy. Consequently, group 2 innate lymphoid cells may be considered as potential therapeutic targets.

Localization of G1A1a Allergenic Domain Destroyed by Thermal Processing.

Glycinin is an important allergenic protein. A1a is the acidic chain of the G1 subunit in glycinin (G1A1a), and it has strong allergenicity. In this study, we used phage display technology to express the protein of G1A1a and its overlapping fragments and an indirect enzyme-linked immunosorbent assay (iELISA) to determine the antigenicity and allergenicity of the expressed protein. After three rounds of screening, it was determined that fragment A1a-2-B-I (151SLENQLDQMPRRFYLAGNQEQEFLKYQQEQG181) is the allergenic domain of G1A1a destroyed by thermal processing. In addition, three overlapping peptides were synthesized from fragments A1a-2-B-I, and a linear epitope was found in this domain through methods including dot blot and iELISA. Peptide 2 (157DQMPRRFYLANGNQE170) showed allergenicity, and after replacing it with alanine, it was found that amino acids D157, Q158, M159, and Y164 were the key amino acids that affected its antigenicity, while Q158, M159, R162, and N168 affected allergenicity.

[In silico analysis of Cit s 2: A highly conserved profilin]. / Análisis in silico de cit s 2: una profilina altamente conservada.

OBJECTIVE: Analyze phylogenetic relationships and molecular mimicry of Cit s 2 and other plant profilins. METHODS: Online bioinformatics tools including Basic Local Alignment Search Tool (BLASTP), PRALINE and MEGA were used for multiple alignments and phylogenetic analysis. A 3D-homology model of Cit s 2 was predicted. Models were calculated with MODELLER. The best model was selected with the model scoring option of MAESTRO. Conserved regions between Cit s 2 and other profilins were located on the 3D model and antigenic regions were predicted by ElliPro server (3-5). RESULTS: Cit s 2 amino acid sequence (Uniprot code:P84177) was compared with other 30 profilins from different allergenic sources. The identity between Cit s 2 and other profilins ranged between 82 and 99%. The highest identity was observed with Cucumis melo (99%) followed by Prunus persica (98%) and Malus domestica (92%). High conserved antigenic regions were observed on the 3D predicted model. Seven lineal and six discontinuous epitopes were found in Cit s 2. CONCLUSION: High conserved antigenic regions were observed on the 3D predicted model of Cit s 2, which might involve potential cross-reactivity between Cit s 2 and other profilins. Future studies are needed to further analyze these results.

OBJETIVO: Analizar las relaciones filogenéticas y el mimetismo molecular de Cit s 2 y otras profilinas vegetales. MÉTODOS: Se utilizaron herramientas bioinformáticas en línea, incluida la de búsqueda de alineación local básica (BLASTP), PRALINE y MEGA, para alineamientos múltiples y análisis filogenético. Se predijo un modelo de homología 3D de Cit s 2. Los modelos se calcularon con MODELLER. El mejor modelo fue seleccionado con la opción de puntuación de modelo de Maestro. Las regiones conservadas entre Cit s 2 y otras profilinas se ubicaron en el modelo 3D y las regiones antigénicas fueron predichas por el servidor ElliPro (3-5). RESULTADOS: La secuencia de aminoácidos de Cit s 2 (código Uniprot: P84177), se comparó con otras 30 profilinas de diferentes fuentes alergénicas. La mayor identidad se observó con Cucumis melo (99%) seguida de Prunus persica (98%) y Malus domestica (92%). Se observaron regiones antigénicas altamente conservadas en el modelo predicho en 3D. Se encontraron siete epítopes lineales, y seis epítopes discontinuos en Cit s 2. CONCLUSIÓN: Se observaron regiones antigénicas altamente conservadas en el modelo 3D predicho de Cit s 2, lo que podría implicar una posible reactividad cruzada entre Cit s 2 y otras profilinas. Se necesitan estudios futuros para analizar más a fondo estos resultados.

Ratiometric Fluorescence Aptasensor of Allergen Protein Based on Multivalent Aptamer-Encoded DNA Flowers as Fluorescence Resonance Energy Transfer Platform.

Life-threatening allergic reactions to food allergens, particularly peanut protein Ara h1, are a growing public health concern affecting millions of people worldwide. Thus, accurate and rapid detection is necessary for allergen labeling and dietary guidance and ultimately preventing allergic incidents. Herein, we present a novel ratiometric fluorescence aptasensor based on multivalent aptamer-encoded DNA flowers (Mul-DNFs) for the high-stability and sensitive detection of allergen Ara h1. The flower-shaped Mul-DNFs were spontaneously packaged using ultralong polymeric DNA amplicons driven by a rolling circle amplification reaction, which contains a large number of Ara h1 specific recognition units and has excellent binding properties. Furthermore, dual-color fluorescence-labeled Mul-DNFs probes were developed by hybridizing them with Cy3- and Cy5-labeled complementary DNA (cDNA) to serve as a ratiometric fluorescence aptasensor platform based on fluorescence resonance energy transfer. Benefiting from the combined merits of the extraordinary synergistic multivalent binding ability of Mul-DNFs, the excellent specificity of the aptamer, and the sensitivity of the ratiometric sensor to avoid exogenous interference. The developed ratiometric aptasensor showed excellent linearity (0.05-2000 ng mL-1) with a limit of detection of 0.02 ng mL-1. Additionally, the developed ratiometric fluorescence aptasensor was utilized for quantifying the presence of Ara h1 in milk, infant milk powder, cookies, bread, and chocolate with recoveries of 95.7-106.3%. The proposed ratiometric aptasensor is expected to be a prospective universal aptasensor platform for the rapid, sensitive, and accurate determination of food and environmental hazards.

Liposomes decorated with ß-conglycinin and glycinin: Construction, structure and in vitro digestive stability.

Liposomes were modified with different proportions of ß-conglycinin (7S) and glycinin (11S) to form Lip-7S and Lip-11S. The morphology, interaction and in vitro simulated digestion of liposomes were studied. The particle size of Lip-7S was smaller than that of Lip-11S. When the values of Lip-7S and Lip-11S were 1:1 and 1:0.75, respectively, the ζ-potential had the maximum absolute value and the dispersion of the system was good. The results of multispectral analysis showed that hydrogen-bond and hydrophobic interaction dominated protein-modified liposomes, the protein structure adsorbed on the surface of liposomes changed, the content of α-helix decreased, and the structure of protein-modified liposomes became denser. The surface hydrophobicity and micropolarity of liposomes decreased with the increase of protein ratio, and tended to be stable after Lip-7S (1:1) and Lip-11S (1:0.75). Differential scanning calorimetry showed that Lip-7S had higher phase transition temperature (≥170.5 °C) and better rigid structure. During simulated digestion, Lip-7S (22.5 %) released less Morin than Lip (40.6 %) and Lip-11S (26.2 %), and effectively delayed the release of FFAs. The environmental stability of liposomes was effectively improved by protein modification, and 7S had better modification effect than 11S. This provides a theoretical basis for 7S and 11S modified liposomes, and also provides a data reference for searching for new materials for stabilization of liposomes.

Neutralizing IgG4 antibodies are a biomarker of sustained efficacy after peanut oral immunotherapy.

BACKGROUND: Clinical efficacy of oral immunotherapy (OIT) has been associated with the induction of blocking antibodies, particularly those capable of disrupting IgE-allergen interactions. Previously, we identified mAbs to Ara h 2 and structurally characterized their epitopes. OBJECTIVE: We investigated longitudinal changes during OIT in antibody binding to conformational epitopes and correlated the results with isotype and clinical efficacy. METHODS: We developed an indirect inhibitory ELISA using mAbs to block conformational epitopes on immobilized Ara h 2 from binding to serum immunoglobulins from peanut-allergic patients undergoing OIT. We tested the functional blocking ability of mAbs using passive cutaneous anaphylaxis in mice with humanized FcεRI receptors. RESULTS: Diverse serum IgE recognition of Ara h 2 conformational epitopes are similar before and after OIT. Optimal inhibition of serum IgE occurs with the combination of 2 neutralizing mAbs (nAbs) recognizing epitopes 1.2 and 3, compared to 2 nonneutralizing mAbs (non-nAbs). After OIT, IgG4 nAbs, but not IgG1 or IgG2 nAbs, increased in sustained compared to transient outcomes. Induction of IgG4 nAbs occurs after OIT only in those with sustained efficacy. Murine passive cutaneous anaphylaxis after sensitization with pooled human sera is significantly inhibited by nAbs compared to non-nAbs. CONCLUSIONS: Serum IgE conformational epitope diversity remains unchanged during OIT. However, IgG4 nAbs capable of uniquely disrupting IgE-allergen interactions to prevent effector cell activation are selectively induced in OIT-treated individuals with sustained clinical efficacy. Therefore, the induction of neutralizing IgG4 antibodies to Ara h 2 are clinically relevant biomarkers of durable efficacy in OIT.