Molecular cloning of plane pollen allergen Pla a 3 and its utility as diagnostic marker for peach associated plane pollen allergy.

BACKGROUND: Non-specific lipid transfer proteins (nsLTP) are considered to provoke allergic symptoms to plane tree pollen, which are frequently associated with peach allergy. OBJECTIVE: The objective was to clone the cDNA of plane pollen nsLTP Pla a 3, to characterize IgE-binding and allergenic potency of recombinant Pla a 3 in comparison to its natural counterpart and peach nsLTP Pru p 3. METHODS: Natural Pla a 3 was purified from plane pollen and analysed by mass spectrometry (MS). Recombinant Pla a 3 was characterized by SDS-PAGE and CD spectroscopy. Specific IgE to extract, components of plane pollen and Pru p 3 was measured by ImmunoCAP in sera of patients allergic to either plane pollen (n = 10), peach (n = 15) or both (n = 15). Biological potency of the proteins was investigated by in vitro mediator release assays and IgE cross-reactivity by competitive ELISA. RESULTS: Two Pla a 3 isoforms were identified. Recombinant Pla a 3 showed high purity, structural integrity, IgE-binding capacity comparable to nPla a 3 and biological potency. Sensitization to plane pollen extract was confirmed in 24/25 plane pollen allergics. The frequency of sensitization to Pla a 3 was 53% among patients allergic to both plane pollen and peach and 10% among plane pollen allergics tolerating peach where most patients were sensitized to Pla a 1. Pla a 3 and Pru p 3 showed strong bi-directional IgE cross-reactivity in patients allergic to peach and plane pollen, but not in peach allergics tolerating plane pollen. Levels of IgE-binding were generally higher to Pru p 3 than to Pla a 3. CONCLUSION: Sensitization to Pla a 3 is relevant in a subgroup of plane pollen allergics with concomitant peach allergy. IgE testing with Pla a 3 may serve as a marker to identify plane pollen allergic patients at risk of LTP-mediated food reactions and thereby improve in vitro diagnostic procedures.

Immunoglobulin E reactivity and allergenic potency of Morus papyrifera (paper mulberry) pollen.

BACKGROUND: Paper mulberry (Morus papyrifera) pollen is considered to be one of the most clinically relevant aeroallergens in Pakistan. To date, the allergenicity of the pollen has not been investigated. OBJECTIVE: To characterize the sensitization profile of mulberry-allergic patients and the proteins of paper mulberry pollen contributing to pollinosis in the Pakistani population. METHODS: Proteins were extracted from mulberry pollen using different protocols. Immunoglobulin (Ig) E binding proteins to mulberry pollen was determined by ImmunoCAP testing and immunoblotting using sera from 29 mulberry pollen-allergic patients with positive skin prick test results to mulberry pollen antigens. The histamine release assay was performed in vitro to determine the allergenic potency of pollen extracts and a partially purified mulberry pollen allergen. The protein was identified using N-terminal sequencing and matrix-assisted laser desorption/ionization-time of flight spectrometry (MALDI-TOF/TOF). RESULTS: IgE sensitization to mulberry pollen was confirmed by positive ImmunoCAP results to pollen from Morus alba (white mulberry) in 23 out of 29 mulberry pollen-allergic patients. A 10-kDa protein from the paper mulberry pollen extract was considered a major allergen, along with additional IgE-reactive proteins. Sera from 79% of the patients reacted to the 10-kDa allergen, which showed substantial capacity to trigger histamine release in 3 out of 4 patients. N-terminal sequencing and MALDI-TOF/TOF yielded an amino acid sequence with no homology to known proteins. CONCLUSIONS: Mulberry-allergic patients are sensitized to multiple mulberry pollen allergens. We identified a novel 10-kDa protein that was a major allergen and should be further investigated for diagnostic and therapeutic purposes.

Immunoglobulin E Reactivity and Allergenic Potency of Morus papyrifera (Paper Mulberry) Pollen

Antecedentes: El polen de la morera del papel se considera uno de los aeroalérgenos más relevantes en Pakistán, cuyas propiedades alergénicas no han sido estudiadas hasta el momento actual. Objetivo: El objetivo de este estudio fue caracterizar el perfil de sensibilización de los pacientes alérgicos a las proteínas de este polen que contribuye a la polinosis en Pakistán. Métodos: La extracción de las proteínas de este polen fue realizada mediante diferentes protocolos. La unión de la IgE a proteínas del polen de la morera del papel, perteneciente a la familia de las moráceas fue determinada mediante InmunoCAP e Inmunoblotting utilizando suero de 29 pacientes alérgicos a este polen con prueba cutánea positiva. Se realizó test de liberación de histamina in vitro para determinar la potencia alergénica de los extractos de polen y de un alérgeno parcialmente purificado. Se secuenciaron la N-terminal y MALDI-TOF/TOF para identificar la proteína. Resultados: En cuanto a los resultados obtenidos se confirmó la sensibilización a dicho polen mediante ImmunoCAP frente a polen de Morus alba en 23 de los 29 pacientes alérgicos al polen de morera del papel. Una proteína de 10 kDa del extracto de dicho polen se consideró como el alérgeno mayor sobre el resto de las proteínas reactivas a la IgE. El suero del 79% de los pacientes reaccionó con este alérgeno de 10 kDa, el cual mostró capacidad para liberar histamina in vitro en 3 de 4 pacientes. La secuenciación N-terminal y MALDI-TOF/TOF arrojó una secuencia de aminoácidos con ausencia de homología con otras proteínas conocidas. Conclusiones: En conclusión, los pacientes alérgicos al polen de morera del papel están sensibilizados a múltiples alérgenos de este polen. Se identifica una nueva proteína de 10 kDa como alérgeno mayoritario que deberá ser investigado con fines diagnósticos y terapéuticos (AU)

Background: Paper mulberry (Morus papyrifera) pollen is considered to be one of the most clinically relevant aeroallergens in Pakistan. To date, the allergenicity of the pollen has not been investigated. Objective: To characterize the sensitization profile of mulberry-allergic patients and the proteins of paper mulberry pollen contributing to pollinosis in the Pakistani population. Methods: Proteins were extracted from mulberry pollen using different protocols. Immunoglobulin (Ig) E binding proteins to mulberry pollen was determined by ImmunoCAP testing and immunoblotting using sera from 29 mulberry pollen–allergic patients with positive skin prick test results to mulberry pollen antigens. The histamine release assay was performed in vitro to determine the allergenic potency of pollen extracts and a partially purified mulberry pollen allergen. The protein was identified using N-terminal sequencing and matrix-assisted laser desorption/ionization–time of flight spectrometry (MALDI-TOF/TOF). Results: IgE sensitization to mulberry pollen was confirmed by positive ImmunoCAP results to pollen from Morus alba (white mulberry) in 23 out of 29 mulberry pollen–allergic patients. A 10-kDa protein from the paper mulberry pollen extract was considered a major allergen, along with additional IgE-reactive proteins. Sera from 79% of the patients reacted to the 10-kDa allergen, which showed substantial capacity to trigger histamine release in 3 out of 4 patients. N-terminal sequencing and MALDI-TOF/TOF yielded an amino acid sequence with no homology to known proteins. Conclusions: Mulberry-allergic patients are sensitized to multiple mulberry pollen allergens. We identified a novel 10-kDa protein that was a major allergen and should be further investigated for diagnostic and therapeutic purposes (AU)

Component-resolved in vitro diagnosis in carrot allergy: does the use of recombinant carrot allergens improve the reliability of the diagnostic procedure?

BACKGROUND: In Europe, pollen-related food allergy is the most frequent form of food allergy in adults. Reliability of current diagnostic procedures, however, is poor and therapeutic options are not available. OBJECTIVES: In the present study, we created a panel of recombinant allergens from carrot and evaluated its potential in component-resolved in vitro diagnosis of carrot allergy. METHODS: Recombinant (r) Dau c 1.0104, Dau c 1.0201 and Dau c 4 were cloned by a polymerase chain reaction strategy, expressed in Escherichia coli and purified. Carrot lipid transfer protein (LTP) was expressed in the yeast Pichia pastoris. Sera from 40 carrot-allergic patients were investigated. Twenty-one birch pollen-allergic subjects with negative open provocation to carrot and 20 non-allergic subjects were included as controls. IgE binding to recombinant allergens as well as to cross-reactive carbohydrate determinants (CCD) was measured by ELISA. Cross-reactivity between Dau c 1 isoforms and Bet v 1 was assayed by ELISA inhibition. Biological activity of the recombinant carrot allergens was assessed by histamine release assay and peripheral blood mononuclear cells stimulation. RESULTS: Ninety-eight percent of the carrot-allergic patients were positive to at least one recombinant allergen; 98% reacted to rDau c 1.0104, 65% to rDau c 1.0201, 38% to rDau c 4 and 20% had IgE against CCD. Specificity using the recombinant allergens was high when compared with non-allergic controls, but low compared with birch-sensitized subjects without carrot allergy. Sensitization to Dau c 1.0201, however, proved to be highly specific for clinically relevant sensitization. Inhibition assays indicated the absence of LTP in carrot root extract, and epitope diversity between Dau c 1.0104, Dau c 1.0201 and Bet v 1. CONCLUSIONS: Our panel of recombinant allergens from carrot can provide a standardized tool for in vitro diagnosis of carrot allergy, and for epitope studies.

IgE reactivity to profilin in pollen-sensitized subjects with adverse reactions to banana and pineapple.

BACKGROUND: The so-called 'latex-fruit syndrome' is a well-documented phenomenon in cross-reactive allergies. By contrast, there is a lack of information about allergy to exotic fruits in patients with a predominant pollen sensitization. Since the ubiquitous protein profilin has been identified as an allergen in natural rubber latex as well as in pollen-related foods, the aim of this study was to investigate the role of profilin in allergy to certain exotic fruits. METHODS: Recombinant profilins from banana and pineapple were cloned by a PCR technique after isolation of total RNA using degenerated profilin-specific primers. The unknown 5' ends of copy DNA (cDNA) were identified by rapid amplification of 5'cDNA ends (5'-RACE) and expression in Escherichia coli BL21(DE3) cells. The recombinant profilins were purified by affinity chromatography using poly-(L)-proline as the solid phase. IgE-binding capabilities were characterized by means of immunoblot and Enzyme Allergosorbent Test (EAST). The cross-reactivity to birch pollen profilin and latex profilin was studied by EAST as well as by immunoblot inhibition experiments. RESULTS: Both banana and pineapple profilin were found to consist of 131 amino acid residues with high amino acid sequence identity to known allergenic pollen and food profilins (71-84%). IgE binding to the recombinant profilins was observed in 7/16 sera from subjects with suspected banana allergy (44%) and in 8/19 sera from subjects with suspected pineapple allergy (42%). Inhibition experiments indicated similar IgE reactivity of natural and recombinant allergens. In addition, high cross-reactivity to birch pollen profilin Bet v 2 and latex profilin Hev b 8 was demonstrated by immunoblot inhibition as well as EAST inhibition experiments. CONCLUSIONS: Since a high IgE-binding prevalence of about 40% was obtained in both banana and pineapple allergy, we conclude that profilin is an important mediator of IgE cross-reactivity between pollen and exotic fruits.

Phenylcoumaran benzylic ether and isoflavonoid reductases are a new class of cross-reactive allergens in birch pollen, fruits and vegetables.

We investigated the biochemical function of the birch pollen allergen Bet v 6 and its role in the IgE-cross-reactivity between birch pollen and plant foods, and characterized Pyr c 5, a Bet v 6-related food allergen, from pear; the proteins were expressed as His-Tag fusion proteins in Eschershia coli and purified by Ni-chelate affinity chromatography under native conditions. Nonfusion proteins were obtained by factor Xa protease treatment. The highest degree of amino-acid sequence identity of Pyr c 5 and Bet v 6 was found with a plant protein related to a defense mechanism, which we have named phenylcoumaran benzylic ether reductase (PCBER) based on its ability to catalyze the NADPH-dependent reduction of 8-5' linked lignans such as dehydrodiconiferyl alcohol to give isodihydrodehydrodiconiferyl alcohol. Enzymatic assays with recombinant Pyr c 5 and Bet v 6 showed PCBER catalytic activity for both recombinant allergens. Both Pyr c 5 and Bet v 6 allergens had similar IgE binding characteristics in immunoblotting and enzyme allergosorbent tests (EAST), and bound IgE from 10 sera of birch-pollen-allergic patients including six pear-allergic subjects. EAST inhibition experiments with Pyr c 5 as the solid phase antigen suggested that homologous allergens may be present in many vegetable foods such as apple, peach, orange, lychee fruit, strawberry, persimmon, zucchini (courgette), and carrot. In extracts of pear, apple, orange, and persimmon, the presence of proteins of approximately 30-35 kDa containing Bet v 6 cross-reactive epitopes was demonstrated with two Bet v 6-specific monoclonal antibodies. Recombinant Pyr c 5 triggered a strong, dose-dependent mediator release from basophils of a pear-allergic subject, suggesting that Pyr c 5 has the potential to elicit type I allergic reactions.

Carrot allergy: double-blinded, placebo-controlled food challenge and identification of allergens.

BACKGROUND: Allergic reactions to carrot affect up to 25% of food-allergic subjects. Clinical manifestations of carrot allergy and IgE responses to carrot proteins, however, have never been studied in subjects with carrot allergy confirmed by means of double-blinded, placebo-controlled food challenge (DBPCFC). OBJECTIVE: The purposes of this investigation were to confirm clinically relevant sensitizations to carrot by means of DBPCFC, to validate current diagnostic methods, and to identify IgE-reactive carrot proteins in patients with true allergy. METHODS: DBPCFCs were performed in 26 subjects with histories of allergic reactions to carrot. Patients underwent skin prick tests with carrot extract, fresh carrot, and various pollen extracts. Specific IgE to carrot, celery, birch, and mugwort pollen and to rBet v 1, rBet v 2, and rBet v 6 were measured through use of the CAP method. Carrot allergens were identified by means of immunoblotting and blotting inhibition. RESULTS: Twenty of 26 patients had positive DBPCFC results. The sensitivity of the determination of carrot-specific IgE antibodies through use of the CAP method (> or =0.7 kU/L) was 90%, the sensitivity for skin prick testing with commercial extracts was 26%, and the sensitivity for prick-to-prick tests with raw carrot was 100%. The Bet v 1--related major carrot allergen Dau c 1 was recognized by IgE from 85% of patients; 45% were sensitized to cross-reactive carbohydrate determinants and 20% to carrot profilin. In 1 subject, a Bet v 6--related carrot allergen was recognized. In 4 patients, IgE binding to Dau c 1 was not inhibited or was weakly inhibited by rBet v 1 or birch pollen extract. CONCLUSION: This study confirmed the allergenicity of carrot by means of DBPCFC. DBPCFC-positive patients had exclusively specific IgE antibodies to birch pollen--related carrot allergens, Dau c 1 being the major allergen. The lack of inhibition of IgE binding to Dau c 1 by birch allergens in a subgroup of patients might indicate an secondary immune response to new epitopes on the food allergen that are not cross-reactive with Bet v 1.

Cloning of the minor allergen Api g 4 profilin from celery (Apium graveolens) and its cross-reactivity with birch pollen profilin Bet v 2.

BACKGROUND: Profilin is a panallergen that is recognized by IgE from about 20% of birch pollen- and plant food-allergic patients. A subgroup of celery-allergic patients shows IgE-reactivity with this minor allergen. To investigate the IgE-binding potential and cross-reactivity of celery profilin at the molecular level, this study was aimed at the cloning and immunological characterization of this allergen. OBJECTIVES: Cloning, expression and purification of profilin from celery tuber to characterize its immunological properties and its cross-reactivity with birch pollen profilin. METHODS: Cloning of celery profilin was performed by polymerase chain reaction using degenerated primers and a 5'RACE method for the identification of the unknown 5'-end of the cDNA. Expression was carried out in Escherichia coli BL21 (DE3) using a modified vector pET-30a. The recombinant profilin was purified by affinity chromatography on poly L-proline coupled to sepharose. Immunological characterization was performed by immunoblotting, EAST and IgE-inhibition experiments. RESULTS: The coding region of the cDNA of celery profilin was identified as a 399-bp open reading frame, coding for a protein of 133 amino acids with a calculated molecular weight of 14.3 kDa. The deduced amino acid sequence of the corresponding protein showed high identity with other plant profilins (71-82%) recently described as allergens. Celery profilin was isolated as highly pure nonfusion protein. The IgE-reactivity of celery profilin was similar to that of natural protein. Seven of 17 celery-allergic patients tested presented specific IgE-antibodies to the recombinant protein tested by immunoblotting. Inhibition experiments showed high cross-reactivity of IgE with both profilins from celery and birch pollen. Moreover, the biological activity of recombinant celery profilin was demonstrated by a histamine release assay. CONCLUSIONS: Celery profilin is an important allergenic compound in celery and shows high homology to birch pollen profilin, Bet v 2. According to the revised IUIS allergen nomenclature, we suggest naming the celery profilin Api g 4. In addition to the cross-reacting major allergens Api g 1 and Bet v 1, birch pollinosis and associated allergies to celery can therefore additionally be explained by the cross-reactivity between homologous profilins. Moreover, recombinant Api g 4 may be used for target-specific diagnosis and structural analyses.