Identification of a vicilin-like major allergen from Prosopis juliflora exhibiting cross- reactivity with legume food allergens.

BACKGROUND: Prosopis juliflora is a clinically relevant allergic sensitizer worldwide and shares cross-reactivity with allergens from several tree pollen and food. The present study aims to purify and immunobiochemically characterize a major allergen from Prosopis pollen. The allergen was further investigated for its cross-reactivity with legume allergens. METHODS: Prosopis extract was fractionated by Q Sepharose and Superdex 75 gel filtration column to purify the allergen. Specific IgE against purified protein was estimated via ELISA and immunoblot. The protein was subjected to mass spectrometric analysis. Glycan characterization was performed by Schiff staining and lectin binding assay followed by deglycosylation studies. The functional activity of the purified protein was evaluated by the basophil activation test. Cross-reactivity was assessed by inhibition studies with legume extracts. RESULTS: A 35 kDa protein was purified and showed 75% IgE reactivity with the patients' sera by ELISA and immunoblot. Glycan characterization of protein demonstrated the presence of terminal glucose and mannose residues. A reduction of 40% and 27% in IgE binding was observed upon chemical and enzymatic deglycosylation of the protein, respectively. The glycoprotein allergen upregulates the expression of CD203c on basophils which was significantly reduced upon deglycosylation, signifying its biological ability to activate the effector cells. The identified protein shared significant homology with Lup an 1 from the lupine bean. Immunoblot inhibition studies of the purified allergen with legume extracts underlined high cross-reactive potential. Complete inhibition was observed with peanut and common bean, while up to 70% inhibition was demonstrated with soy, black gram, chickpea, and lima bean. CONCLUSION: A 35 kDa vicilin-like major allergen was isolated from P. juliflora. The protein possesses glycan moieties crucial for IgE binding and basophil activation. Furthermore, the purified protein shows homology with Lup an 1 and exhibits cross-reactivity with common edible legume proteins.

Molecular Cloning and Expression of Pro J 1: A New Allergen of Prosopis Juliflora Pollen.

Pollen from mesquite (Prosopis juliflora) is one of the important causes of immediate hypersensitivity reactions in the arid and semi-arid regions of the world. The aim of present study is to produce and purify the recombinant form of allergenic Ole e 1-like protein from the pollen of this allergenic tree. Immunological and cross-inhibition assays were performed for the evaluation of IgE-binding capacity of purified recombinant protein. For molecular cloning, the coding sequence of the mesquite Ole e 1-like protein was inserted into pTZ57R/T vector and expressed in Escherichia coli using the vector pET-21b(+). After purification of the recombinant protein, its immunoreactivity was analysed by in vitro assays using sera from twenty one patients with an allergy to mesquite pollen. The purified recombinant allergen was a member of Ole e 1-like protein family and consisted of 150 amino acid residues, with a predicted molecular mass of 16.5 kDa and a calculated isoelectric point (pI) of 4.75. Twelve patients (57.14%) had significant specific IgE levels for this recombinant allergen. Immunodetection and inhibition assays indicated that the purified recombinant allergen might be the same as that in the crude extract. Herein, we introduce an important new allergen from P. juliflora pollen (Pro j 1), which is a member of the Ole e 1-like protein family and exhibits significant identity and similarity to other allergenic members of this family.

Broad bean-induced anaphylaxis: a case report

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Pro j 2 is mesquite profilin: molecular characteristics and specific IgE binding activity.

BACKGROUND: Pollens from mesquite (Prosopis juliflora) are potent allergen responsible in causing immediate hypersensitivity reactions in susceptible people in tropical countries. OBJECTIVE: This study aimed to clone, express and purify the mesquite pollen profilin (Pro j 2) as well as evaluating its nucleotide sequence homology in order to predict allergenic cross-reactivity with profilins of common allergenic plants. METHODS: Immunoblotting assay and specific ELISA were applied to determine the immunoreactivity of sera from 35 patients who were allergic to mesquite pollen. The mesquite profilin-coding sequence was cloned into PTZ57R/T vector and amplified. The cDNA of mesquite pollen profilin was then expressed in Escherichia coli using pET-21b (+) vector and puri?ed by one-step Ni2+ a?nity chromatography. IgE binding capacity of the recombinant mesquite profiling (rPro j 2) was analyzed by specific ELISA, immunoblotting, and inhibition assays. RESULTS: cDNA nucleotide sequencing revealed an open reading frame of 399bp encoding for 133 amino acids which belongs to the profilin family. Seventeen patients (17/35, 48.57%) had significant specific IgE level for rPro j 2. Immunodetection and inhibition assays indicated that puri?ed rPro j 2 might be similar as that in the crude extract. CONCLUSION: Pro j 2, as a new allergen from mesquite pollen, was produced in E. coli with an IgE-reactivity similar to that of its natural counterpart. The amino acid sequences homology analysis of mesquite profilin and several profilin molecules from other plants showed high degree of cross-reactivity among plant-derived profilins from unrelated families.

Immunochemical characterization of prosopis juliflora pollen allergens and evaluation of cross-reactivity pattern with the most allergenic pollens in tropical areas.

Allergy to Prosopis juliflora (mesquite) pollen is one of the common causes of respiratory allergy in tropical countries. Mesquite is widely used as street trees in towns and ornamental shade trees in parks and gardens throughout arid and semiarid regions of Iran. The inhalation of mesquite pollen and several species of Amaranthus/Chenopodiaceae family is the most important cause of allergic respiratory symptoms in Khuzestan province. This study was designed to evaluate IgE banding proteins of mesquite pollen extract and its IgE cross-reactivity with other allergenic plants. Twenty patients with allergic symptoms and positive skin prick tests (SPT) for mesquite pollen extract participated in the study. Crude pollen extract was prepared from local mesquite trees and used for the evaluation of allergenic profiles of P. juliflora pollen extract by Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and IgE-immunoblotting. There were several protein bands in mesquite pollen extract using SDS-PAGE with the approximate range of molecular weight of 10-85 kDa. The most frequent IgE reactive bands among the patients' sera were approximately 20 and 66 kDa. However, there were other IgE reactive protein bands among the patients' sera with molecular weights of 10, 15, 35, 45, 55 and 85 kDa. Inhibition experiments revealed high IgE cross-reactivity between mesquite and acacia. There are several IgE-binding proteins in P. juliflora pollen extract. Results of this study indicate that proteins with a molecular weight of 10 to 85 kDa are the major allergens in P. juliflora pollen extract.

A clinically relevant major cross-reactive allergen from mesquite tree pollen.

BACKGROUND: Prosopis juliflora (mesquite) is one of the major sources of pollinosis in tropical and semi-arid countries of the world. The present study was undertaken to purify and characterize a major cross-reactive allergen from this tree species. MATERIALS AND METHODS: Mesquite pollen extract was purified using reverse-phase chromatography. Allergen characterization was done by electrophoresis, enzyme-linked immunosorbent assay (ELISA) and Western blotting. Clinical relevance of the purified protein was analyzed by in vivo (skin tests) and in vitro experiments such as ELISA, histamine release, peripheral blood mononuclear cells (PBMC) proliferation and cytokine assays. Cross-reactivity of purified protein with allergenic tree species and lima bean (food) was assessed by inhibition assays. RESULTS: A 66-kDa protein was purified from mesquite pollen extract using octadecyl silica resin. Purified protein recognized 90% of mesquite-sensitized patients in skin test and ELISA. It induced significant histamine release in allergic patients' blood and interleukin-4 secretion in the PBMC culture supernatants. Inhibition assays suggested close allergenic relationship of this protein with Ailanthus excelsa, Cassia siamea, Salvadora persica pollen and Phaseolus lunatus (lima bean - an edible legume). CONCLUSIONS: A 66-kDa major cross-reactive allergen was isolated from mesquite pollen using single-step purification procedure. The protein seems relevant for clinical applications in allergic disorders.

Immunoglobulin E (IgE)-mediated cross-reactivity between mesquite pollen proteins and lima bean, an edible legume.

Immunoglobulin E (IgE)-mediated food allergy often develops as a consequence of allergic sensitization to pollen proteins. Mesquite (Prosopis juliflora) tree pollen is reported to be cross-reactive with other pollen species, but little has been reported on its cross-reactivity with plant-derived foods belonging to the same/different families. The present study investigates the in vitro cross-reactivity of mesquite pollen and lima bean (Phaseolus lunatus), an edible seed belonging to the Leguminosae family. Of 110 patients (asthma, rhinitis or both) tested intradermally, 20 showed marked positive reactions with Prosopis pollen extract. Of these, 12 patients showed elevated specific IgE to Prosopis pollen extract alone and four to both Phaseolus and pollen extract. In vitro cross-reactivity was investigated using inhibition assays [enzyme-linked immunosorbent assay (ELISA) inhibition, immunoblot inhibition], histamine release and lymphoproliferation. P. lunatus extract could inhibit IgE binding to P. juliflora in a dose-dependent manner, requiring 400 ng of protein for 50% inhibition in ELISA assay. Immunoblot and immunoblot inhibition demonstrated the presence of 20, 26, 35, 66 and 72 kDa as shared IgE binding components between the two extracts. Histamine release, peripheral blood mononuclear cells proliferation and interleukin (IL)-4 levels also suggested allergenic cross-reactivity. In conclusion, there is humoral and cellular cross-reactivity between Prosopis pollen and Phaseolus seed allergens.

Analysis of IgE binding proteins of mesquite (Prosopis juliflora) pollen and cross-reactivity with predominant tree pollens.

Pollen from the mesquite tree, Prosopis juliflora, is an important source of respiratory allergy in tropical countries. Our aim was to partially characterize the IgE binding proteins of P. juliflora pollen extract and study cross-reactivity with prevalent tree pollen allergens. Intradermal tests with P. juliflora and five other tree pollen extracts were performed on respiratory allergy patients from Bikaner (arid) and Delhi (semi arid). Prosopis extract elicited positive skin reactions in 71/220 of the patients. Sera were collected from 38 of these 71 patients and all demonstrated elevated specific IgE to P. juliflora. Immunoblotting with pooled patients' sera demonstrated 16 IgE binding components, with components of 24, 26, 29, 31, 35, 52, 58, 66 and 95 kDa recognized by more than 80% of individual patients' sera. P. juliflora extract is allergenically potent requiring 73 ng of self-protein for 50% inhibition of IgE binding in ELISA inhibition. Cross-inhibition assays showed close relationship among P. juliflora, Ailanthus excelsa, Cassia siamea and Salvadora persica. IgE binding components of 14, 41, 52 and 66 kDa were shared allergens whereas 26 and 29 kDa were specific to P. juliflora. The findings suggest that purification of cross-reactive allergens will be helpful for diagnosis and immunotherapy of tree pollen allergic patients.

Dynamic changes in sensitization to specific aeroallergens in children raised in a desert environment.

BACKGROUND: Allergen skin test reactivity and total serum IgE are objective measures used to characterize and help diagnose allergic diseases. Cross-sectional studies have shown that overall aeroallergen skin test reactivity increases throughout childhood. However, little attention has been paid to whether individual aeroallergen remittance occurs, which could distort or mask relationships to disease. OBJECTIVE: To access the incidence and remittance of skin test reactions to individual allergens in children aged 6-11 years. METHODS: Longitudinal sensitization to six aeroallergens and total IgE were assessed in 828 children raised in the semi-arid US southwest at ages 6 and 11 years. RESULTS: New sensitization (to any allergen) between 6 and 11 years occurred in 30.2% of children compared with 39.7% before age 6 years. The rate of complete remittance from positive to negative between ages 6 and 11 years was 8.2%, and total IgE at age 6 years was not predictive. Remittance rates for individual allergens were high and variable (19-49%). The perennial allergens Bermuda and Alternaria were early sensitizers and had low remittance rates. Early sensitization to the four seasonal allergens was less common and more subject to remittance with the bulk of sensitization occurring between 6 and 11 years. CONCLUSION: This study shows that sensitization to individual aeroallergens in childhood is dynamic and indicates the limitation of single point assessment of skin test reactivity.

Application of antibodies for the identification of polysaccharide gum additives in processed foods.

Anti-carbohydrate antibodies with specificities for polysaccharide gums were isolated from the serum of rabbits that were immunized with a solution of the gums and Freund's complete adjuvant. The primary objective was to test an immunological method for the detection of the polysaccharide gums as additives to processed foods. Analysis involved the extraction of food with phosphate buffer and the testing of the extract for a reaction with anti-gum antibodies by the agar diffusion method. Reaction by a specific gum with the homologous antibodies establishes the presence of the gum in the food. The method is a novel application of antibodies. The antibody method is highly specific for a gum and thus possesses advantages over other methods of analysis for polysaccharide gums as additives in processed foods.