Association Between the Seed Storage Proteins 2S Albumin and 11S Globulin and Severe Allergic Reaction After Flaxseed Intake.

BACKGROUND: Given the increased popularity of flaxseed in meals, several cases of allergy to these seeds have been reported. Little is known about the allergens implicated in hypersensitivity reactions to flaxseed. The present study aimed to identify the allergens involved in IgE-mediated reactions in 5 patients with a clinical history of severe systemic symptoms after flaxseed consumption. METHODS: Proteins that were potential allergens with IgE-binding capacity were purified from flaxseed extract using chromatography and identified via MALDI-TOF mass spectrometry. Immunoassays were performed using the 5 allergic patients' sera tested individually and as a pool. RESULTS: Immunoblotting of the flaxseed extract revealed a low-molecular-mass protein (around 13 kDa) in 4 of the 5 patients, while a protein of approximately 55 kDa was detected in 2 patients. The proteins were identified by mass spectrometry as flaxseed 2S albumin, which is included in the WHO/IUIS allergen nomenclature as Lin u 1, and 11S globulin. Inhibition assays revealed in vitro IgE-mediated cross-reactivity between Lin u 1 and peanut and cashew nut proteins, while IgE-mediated recognition of 11S globulin by patients' sera was partially inhibited by several plant-derived sources. CONCLUSIONS: Seed storage proteins from flaxseed were involved in the development of severe symptoms in the 5 patients studied and exhibited cross-reactivity with other allergenic sources. Besides the severity of flaxseed allergy in patients sensitized to 2S albumin, this is the first time that 11S globulin has been identified as a potential allergen. Taking these data into account should ensure a more accurate diagnosis.

Airway Epithelium Plays a Leading Role in the Complex Framework Underlying Respiratory Allergy.

Airway epithelium is the cellular structure with the greatest surface exposed to a plethora of environmental airborne substances, including microorganisms, respiratory viruses, air pollutants, and allergens. In addition to being a protective physical barrier at the air-liquid interface, the airway epithelium acts as an effective chemical and immunological barrier that plays a crucial role in orchestrating the immune response in the lungs, by supporting the activation, recruitment, and mobilization of immune cells. Airway epithelium dysfunction has been clearly associated with various airway inflammatory diseases, such as allergic asthma. Although it is not fully understood why a person develops respiratory allergy, a growing body of evidence shows that the nature of the host's immune response is strongly determined by the state of the airway epithelium at the time of contact with the inhaled allergen. Our review highlights the physiological state of airway epithelium as a key element in the development of allergy and, particularly, in exacerbation of asthma. We review the role of physiological oxidants as signaling molecules in lung biology and allergic diseases and examine how high exposure to air pollutants (eg, cigarette smoke and diesel particles) can contribute to the increased incidence of respiratory allergy and exacerbation of the disease.

Amaranthaceae pollens: review of an emerging allergy in the mediterranean area.

The Amaranthaceae family is composed of about 180 genera and 2500 species. These common weeds have become increasingly relevant as triggers of allergy in the last few years, as they are able to rapidly colonize salty and arid soils in extensive desert areas. The genera Chenopodium, Salsola, and Amaranthus are the major sources of pollinosis from the Amaranthaceae family in southern Europe, western United States, and semidesert areas of Saudi Arabia, Kuwait, and Iran. In Spain, Salsola kali is one of the most relevant causes of pollinosis, together with olive and grasses. To date, 9Amaranthaceae pollen allergens from Chenopodium album, Salsola kali, and Amaranthus retroflexus have been described and are listed in the International Union of Immunological Societies allergen nomenclature database.The major allergens ofAmaranthaceae pollen belong to the pectin methylesterase, Ole e 1-like, and profilin panallergen families, whereas the minor allergens belong to the cobalamin- independent methionine synthase and polcalcin panallergen families. These relevant allergens have been characterized physicochemically, and immunologically at different levels. Recombinant forms, allergenic fusion recombinant proteins, and hypoallergenic derivatives of these allergens have been expressed in bacteria and yeast and compared with their natural proteins from pollen. In this review, we provide an extensive overview ofAmaranthaceae pollen allergens, focusing on their physicochemical, and immunological properties and on their clinical significance in allergic patients. We also review studies where these recombinant allergens and their hypoallergenic derivatives have been used in clinical diagnosis and their potential use in personalized therapy.

Characterization of profilin and polcalcin panallergens from ash pollen.

BACKGROUND: Ash (Fraxinus excelsior) is an important source of allergenic pollen in temperate areas of Europe. Profilin and polcalcin are 2 important panallergens involved in cross-reactivity between different sources. OBJECTIVE: To clone and produce Fra e 2 (profilin) and Fra e 3 (polcalcin) as recombinant proteins and evaluate their immunological properties using the natural forms obtained from ash pollen. METHODS: Total RNA from ash pollen was used as a template to obtain the specific complementary DNA (cDNA) sequences of the 2 panallergens. The cDNA-encoding sequences were cloned into the pET11b expression vector and used to transform BL21 (DE3) Escherichia coli cells. Proteins were expressed, purified by chromatography, and characterized structurally by circular dichroism, mass spectrometry, and immunologically by western blot and ELISA using profilin and polcalcin polyclonal antibodies and human sera from ash pollen-sensitized patients. RESULTS: Profilin and polcalcin amino acid sequences from ash pollen showed a high degree of identity with homologous allergens from different sources. The cDNA-encoding allergen sequences were expressed as nonfusion recombinant proteins and purified to homogeneity. Secondary structure values were similar to those obtained from other members of these families. Allergenic properties of the recombinant allergens were observed to be equivalent to those of the natural counterparts of F excelsior pollen. CONCLUSIONS: Fra e 2 and Fra e 3 recombinant allergens might be used in clinical diagnosis to determine profilin- and polcalcin-specific IgE levels present in the sera of ash pollen-sensitized patients, thus facilitating the finding of the sensitizing source in areas with complex sensitization profiles.

A deletion variant of the Aspergillus fumigatus ribotoxin Asp f 1 induces an attenuated airway inflammatory response in a mouse model of sensitization.

BACKGROUND: Aspergillus fumigatus is the most prevalent airborne fungal pathogen, and the ribotoxin Asp f 1 is one of its major allergens. Alpha-Sarcin is a natural variant of Asp f 1 produced by the nonpathogenic fungus Aspergillus giganteus. Both proteins show a sequence identity of 87% and almost identical 3-dimensional structures. Alpha-Sarcin delta(7-22) is a deletion mutant that displays reduced immunoglobulin (Ig) E reactivity and is much less cytotoxic than wild-type proteins against human transformed cells. OBJECTIVE: A murine model of sensitization to Asp f 1 was established to test the response elicited by this alpha-sarcin delta(7-22) deletion mutant. METHODS: BALB/c mice were treated intraperitoneally with different mixtures of recombinant wild-type Asp f 1 and/or a suspension of a commercially available A. fumigatus standard extract. Mice were then intranasally challenged with Asp f 1 or alpha-sarcin delta(7-22). Sera were collected for subsequent measurement of Ig levels and histological analysis of the nostrils and lungs. RESULTS: Sensitization to Asp f 1 was successful only when the purified protein was first administered together with the A fumigatus suspension. The model was characterized by elevated levels of total IgE in serum and histological lesions in the lungs and nostrils. These symptoms were less severe when the deletion variant was the protein administered, thus confirming in vivo its lower toxic character. CONCLUSIONS: An easily reproducible mouse model of A fumigatus Asp f 1 sensitization was established. This model revealed alpha-sarcin delta(7-22) to be a potential candidate for immunotherapy.

Intranasal vaccination with poly(lactide-co-glycolide) microparticles containing a peptide T of Ole e 1 prevents mice against sensitization.

BACKGROUND: Biodegradable microparticles, in particular poly(lactide-co-glycolide) (PLGA), have been shown as potential delivery vehicles for intranasal (i.n.) vaccines in animal models. OBJECTIVES: To evaluate whether i.n. administration of PLGA microparticles containing a peptide with the major T cell epitope of Ole e 1, the main allergen of olive pollen, prevented mice from allergic sensitization to the whole protein. METHODS: Peptide-PLGA microparticles were prepared by a solvent evaporation double emulsion method. Microparticles in a size range of 0.8 mum were evaluated for peptide loading and in vitro antigen release. Stability and immunogenicity of the entrapped peptide were retained, as determined by dot blot and ELISA inhibition. BALB/c mice were intranasally treated with peptide-PLGA microparticles for 3 consecutive days, 1 week before sensitization/challenge to Ole e 1. Blood, lungs and spleen were collected and analysed for immune response. Biodistribution of microparticles was investigated using confocal microscopy. RESULTS: I.n. pretreatment of BALB/c mice with peptide-PLGA microparticles before sensitization to Ole e 1 led to a significant inhibition of serum allergen-specific IgE and IgG1 antibody levels, but a marked increase of specific IgG2a antibodies as compared with sham-pretreated mice. Moreover, IL-5 and IL-10 levels in spleen cell cultures were suppressed in peptide-PLGA pretreated mice. The airway histopathologic parameters associated with inflammation were significantly suppressed by the pretreatment. CONCLUSION: These results demonstrate that i.n. immunization with peptide T-PLGA microparticles is effective in preventing subsequent allergic sensitization to Ole e 1. Our data indicate that peptide-PLGA microparticles may be promising candidates for the design of nasal vaccines against allergic diseases in humans.

Olive pollen recombinant allergens: value in diagnosis and immunotherapy.

Olive pollen has a complex allergenic profile, from which more than 10 allergens have been identified and characterized. Some of these belong to well-known protein families and others cannot be included in reported biochemical types. Most of these allergens have been produced by recombinant technology, mainly in Escherichia coli or in Pichia pastoris, and they are good candidates for diagnostic and therapeutic purposes. Diagnosis and immunotherapy of allergy currently use extracts prepared from homogenates of natural sources, which only allow us to detect sensitivity to the complete source. These extracts can be successfully replaced by mixtures with controlled amounts of specific allergenic proteins obtained by recombinant technology in order to define the sensitization profile of individual patients. Recombinant Ole e 1 can be used as a marker for sensitization to Oleaceae. Recombinants Ole e 2 (profilin) and Ole e 3 (polcalcin) can serve as markers of polysensitivity. Finally, recombinant forms of Ole e 6, Ole e 10, and the carboxy-terminal and amino-terminal domains of Ole e 9 would help to detect sensitization to these minority allergens that could be overlooked in the complete olive pollen extract. These recombinant molecules can help provide an accurate diagnosis of sensitivity to individual allergens and, therefore, improve the design of more efficacious allergen-based immunotherapy strategies.

Association Between the Seed Storage Proteins 2S Albumin and 11S Globulin and Severe Allergic Reaction After Flaxseed Intake

Background: Given the increased popularity of flaxseed in meals, several cases of allergy to these seeds have been reported. Little is known about the allergens implicated in hypersensitivity reactions to flaxseed. The present study aimed to identify the allergens involved in IgE-mediated reactions in 5 patients with a clinical history of severe systemic symptoms after flaxseed consumption. Methods: Proteins that were potential allergens with IgE-binding capacity were purified from flaxseed extract using chromatography and identified via MALDI-TOF mass spectrometry. Immunoassays were performed using the 5 allergic patients’ sera tested individually and as a pool. Results: Immunoblotting of the flaxseed extract revealed a low-molecular-mass protein (around 13 kDa) in 4 of the 5 patients, while a protein of approximately 55 kDa was detected in 2 patients. The proteins were identified by mass spectrometry as flaxseed 2S albumin, which is included in the WHO/IUIS allergen nomenclature as Lin u 1, and 11S globulin. Inhibition assays revealed in vitro IgE-mediated cross-reactivity between Lin u 1 and peanut and cashew nut proteins, while IgE-mediated recognition of 11S globulin by patients’ sera was partially inhibited by several plant-derived sources. Conclusions: Seed storage proteins from flaxseed were involved in the development of severe symptoms in the 5 patients studied and exhibited cross-reactivity with other allergenic sources. Besides the severity of flaxseed allergy in patients sensitized to 2S albumin, this is the first time that 11S globulin has been identified as a potential allergen. Taking these data into account should ensure a more accurate diagnosis. (AU)

Antecedentes: Dada la creciente popularidad de la linaza en las comidas, se han notificado varios casos de alergia a estas semillas. La información acerca de los alérgenos implicados en las reacciones de hipersensibilidad a estas semillas es escasa. El presente trabajo pretende identificar los alérgenos implicados en las reacciones mediadas por IgE en cinco pacientes con una historia clínica de síntomas sistémicos graves tras el consumo de linaza. Métodos: Las proteínas susceptibles de ser alérgenos con capacidad de unir IgE se purificaron a partir del extracto de linaza mediante técnicas cromatográficas. Su identificación se realizó mediante espectrometría de masas MALDI-TOF. Se realizaron inmunoensayos con los sueros de los cinco pacientes alérgicos, utilizados de forma individual o como mezclas. Resultados: Cuatro de los cinco pacientes reconocieron una proteína de baja masa molecular (alrededor de 13 kDa) en inmunoensayos con extracto de linaza, mientras que dos pacientes reconocieron una proteína de aproximadamente 55 kDa. Se identificaron por espectrometría de masas como albúmina 2S de linaza, incluida en la nomenclatura de alérgenos de la OMS/IUIS como Lin u 1, y globulina 11S, respectivamente. Los ensayos de inhibición in vitro revelaron la existencia de reactividad cruzada de la Lin u 1 con las proteínas del cacahuete y del anacardo, mientras que el reconocimiento por parte de la IgE de la globulina 11S por parte de los sueros de los pacientes fue parcialmente inhibido por varias fuentes vegetales. Conclusiones: Las proteínas de almacenamiento de las semillas de lino estaban implicadas en el desarrollo de síntomas graves en cinco individuos y mostraron una reactividad cruzada con otras fuentes alergénicas. Además de la gravedad de la alergia a la linaza en los pacientes sensibilizados a la albúmina 2S, es la primera vez que se identifica la globulina 11S como un alérgeno potencial.

Glycosylation site of the major allergen from olive tree pollen. Allergenic implications of the carbohydrate moiety.

The electrophoretic analysis of purified Ole e I, the major allergen from Olea europaea pollen, reveals the presence of two main variants, glycosylated (20.0 kDa) and non-glycosylated (18.5 kDa) components. The glycosylated variant has been identified as a concanavalin A-binding glycoprotein. Its carbohydrate moiety has a molecular mass of about 1.3 kDa (5% weight of the glycosylated allergen), based on mass spectrometry analysis. Enzymatic treatment of native Ole e I with the specific glycosidase PNGase F accounts for an oligosaccharide N-linked to the polypeptide chain. This treatment does not sensibly modify the secondary structure of the protein but diminishes the affinity of the allergen for specific IgE antibodies. Tryptic digestion of Ole e I reveals the presence of a single carbohydrate-containing peptide. This peptide was recognized by the sera of hypersensitive individuals. The amino acid sequence of this peptide is Phe-Lys-Leu-Asn-Thr-Val-Asn-Gly-Thr-Thr-Arg, asparagine at the seventh being the carbohydrate attaching site. The obtained data are discussed in terms of the potential role of the sugar moiety in the allergenic activity of Ole e I.

Purification, amino acid sequence and immunological characterization of Ole e 6, a cysteine-enriched allergen from olive tree pollen.

The Ole e 6 allergen from olive tree pollen has been isolated by combining gel permeation and reverse-phase chromatographies. It is a single and highly acidic (pI 4.2) polypeptide chain protein. Its NH2-terminal amino acid sequence has been determined by Edman degradation. Total RNA from the olive tree pollen was isolated, and a specific cDNA was amplified by the polymerase chain reaction using a degenerate oligonucleotide primer designed according to the NH2-terminal sequence of the protein. The nucleotide sequencing of the cDNA rendered an open reading frame encoding a 50 amino acid polypeptide chain, in which two sets of the sequential motif Cys-X3-Cys-X3-Cys are present. No sequence similarity has been found between this protein and other previously described polypeptides.

Effects of ole e 1 on human bronchial epithelial cells cultured at the air-liquid interface

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Amaranthaceae pollens: review of an emerging allergy in the mediterranean area

The Amaranthaceae family is composed of about 180 genera and 2500 species. These common weeds have become increasingly relevant as triggers of allergy in the last few years, as they are able to rapidly colonize salty and arid soils in extensive desert areas. The genera Chenopodium, Salsola, and Amaranthus are the major sources of pollinosis from the Amaranthaceae family in southern Europe, western United States, and semidesert areas of Saudi Arabia, Kuwait, and Iran. In Spain, Salsola kali is one of the most relevant causes of pollinosis, together with olive and grasses. To date, 9 Amaranthaceae pollen allergens from Chenopodium album, Salsola kali, and Amaranthus retroflexus have been described and are listed in the International Union of Immunological Societies allergen nomenclature database. The major allergens of Amaranthaceae pollen belong to the pectin methylesterase, Ole e 1-like, and profilin panallergen families, whereas the minor allergens belong to the cobalaminindependent methionine synthase and polcalcin panallergen families. These relevant allergens have been characterized physicochemically, and immunologically at different levels. Recombinant forms, allergenic fusion recombinant proteins, and hypoallergenic derivatives of these allergens have been expressed in bacteria and yeast and compared with their natural proteins from pollen. In this review, we provide an extensive overview of Amaranthaceae pollen allergens, focusing on their physicochemical, and immunological properties and on their clinical significance in allergic patients. We also review studies where these recombinant allergens and their hypoallergenic derivatives have been used in clinical diagnosis and their potential use in personalized therapy (AU)

La familia Amaranthaceae se compone de alrededor de 180 géneros y 2500 especies vegetales. En los últimos años, el polen de estas malezas está adquiriendo una relevancia cada vez mayor como inductor de alergia, ya que estas plantas son capaces de colonizar rápidamente los suelos salinos y áridos de zonas desertificadas. El polen de los géneros Chenopodium, Salsola y Amaranthus es el causante del mayor número de casos de polinosis asociados a la familia Amaranthaceae en países del sur de Europa, oeste de Estados Unidos, y en las zonas semi-desérticas de Arabia Saudí, Kuwait o Irán. En España, el polen de Salsola kali es una de las causas más relevantes de polinosis junto con los pólenes de olivo y gramíneas. Hasta la fecha, se han descrito un total de nueve alérgenos del polen de Chenopodium album, Salsola kali y Amaranthus retroflexus, los cuales se han depositado en la base de datos de nomenclatura de alérgenos IUIS. Los alérgenos principales del polen de la familia Amaranthaceae pertenecen a las familias pectin metilesterasa, Ole e 1, o a la familia de panalérgenos -profilina-, mientras que los alérgenos secundarios descritos pertenecen a la familia de panálergenos -polcalcina-, o bien, corresponden a la metionina sintasa independiente de cobalamina. Estos relevantes alérgenos se han caracterizado fisicoquímica e inmunológicamente en mayor o menor profundidad. Las formas recombinantes, y sus variantes recombinantes o derivados hipoalergénicos fusionados a un tag, se han expresado en bacteria o levadura y se ha comparado su funcionalidad con sus correspondientes homólogos naturales presentes en el polen. En esta revisión, ofrecemos una extensa descripción de los alérgenos del polen de la familia Amaranthaceae, centrándonos en sus propiedades físico-químicas e inmunológicas, y en su importancia clínica en los pacientes alérgicos. Por otra parte, también hemos revisado aquellos estudios en donde se han utilizado estos alérgenos recombinantes y sus derivados hipoalergénicos en el diagnóstico clínico, o bien, en donde se describe su potencial uso en la terapia personalizada (AU)

Characterization of profilin and polcalcin panallergens from ash pollen

Background: Ash ( Fraxinus excelsior ) is an important source of allergenic pollen in temperate areas of Europe. Profilin and polcalcin are 2 important panallergens involved in cross-reactivity between different sources. Objective: To clone and produce Fra e 2 (profilin) and Fra e 3 (polcalcin) as recombinant proteins and evaluate their immunological properties using the natural forms obtained from ash pollen. Methods: Total RNA from ash pollen was used as a template to obtain the specific complementary DNA (cDNA) sequences of the 2 panallergens. The cDNA-encoding sequences were cloned into the pET11b expression vector and used to transform BL21 (DE3) Escherichia coli cells. Proteins were expressed, purified by chromatography, and characterized structurally by circular dichroism, mass spectrometry, and immunologically by western blot and ELISA using profilin and polcalcin polyclonal antibodies and human sera from ash pollen-sensitized patients. Results: Profilin and polcalcin amino acid sequences from ash pollen showed a high degree of identity with homologous allergens from different sources. The cDNA-encoding allergen sequences were expressed as nonfusion recombinant proteins and purified to homogeneity. Secondary structure values were similar to those obtained from other members of these families. Allergenic properties of the recombinant allergens were observed to be equivalent to those of the natural counterparts of F excelsior pollen. Conclusions: Fra e 2 and Fra e 3 recombinant allergens might be used in clinical diagnosis to determine profilin- and polcalcin-specific IgE levels present in the sera of ash pollen-sensitized patients, thus facilitating the finding of the sensitizing source in areas with complex sensitization profiles (AU)

Antecedentes: El polen de fresno (Fraxinus excelsior ) es una importante fuente alergénica en zonas cálidas de Europa. La profilina y polcalcina son 2 panalérgenos implicados en reactividad cruzada. Objetivos: Clonar y producir Fra e 2 (profilina) y Fra e 3 (polcalcina) como alérgenos recombinantes. Comparar sus propiedades inmunológicas con sus formas naturales del polen de fresno. Métodos: El RNA total de polen de fresno se utilizó como molde para obtener los cDNAs específicos de ambos panalérgenos. Dichos cDNAs se clonaron en el vector de expresión pET11b y se transformaron células de Escherichia coli BL21(DE3). Las proteínas se caracterizaron mediante dicroísmo circular, espectrometría de masas, inmunodetección en membrana y ELISA utilizando anticuerpos policlonales frente a profilina y polcalcina y sueros de pacientes alérgicos al polen de fresno. Resultados: Las secuencias de aminoácidos de la profilina y polcalcina de polen de fresno presentaban una identidad de secuencia elevada con alérgenos homólogos. Dichos alérgenos se expresaron como proteínas recombinantes independientes y se purificaron a homogeneidad. Los valores de estructura secundaria fueron similares a los de otros miembros de estas familias. Las propiedades alergénicas de los alérgenos recombinantes resultaron ser equivalentes a los de sus homólogos naturales del polen. Conclusiones: Los alérgenos recombinantes Fra e 2 y Fra e 3 podrían usarse en diagnóstico clínico para determinar los niveles de IgE específicos para profilina y polcalcina en los sueros de los pacientes sensibilizados al polen de fresno, facilitando así la identificación de la fuente sensibilizante en áreas donde los pacientes presentan perfiles alergénicos complejos (AU)

Hypoallergenic mutants of Ole e 1, the major olive pollen allergen, as candidates for allergy vaccines.

BACKGROUND: The C-terminal region of Ole e 1, a major allergen from olive pollen, is a dominant IgE-reactive site and offers a target for site-directed mutagenesis to produce variants with reduced IgE-binding capability. OBJECTIVE: To evaluate in vitro and in vivo the immunogenic properties of three engineered derivatives of Ole e 1. METHODS: One point (Y141A) and two deletion (135Delta10 and 140Delta5) mutants were generated by site-directed mutagenesis of Ole e 1-specific cDNA and produced in Pichia pastoris. Ole e 1 mutants were analysed for IgE reactivity by ELISA using sera from olive pollen-allergic patients. Their allergenicity was also investigated in both a mouse model of allergic sensitization and in basophil activation assays. IgG1 response was assayed by immunoblotting and competitive ELISA. T cell reactivity was evaluated by proliferation assays and cytokine production in splenocyte cultures. RESULTS: The 135Delta10 mutant showed the strongest reduction in the IgE-binding capability of sera from olive pollen-allergic patients. Rat basophil leukaemia assays identified the deletion mutant 135Delta10 as the variant with the lowest beta-hexosaminidase-releasing capacity. Furthermore, the same 135Delta10 mutant induced the lowest IgE levels in a BALB/c mouse model of sensitization. All Ole e 1 mutants retained their allergen-specific T cell reactivity. Immunization of mice with the mutants induced IgG1 antibodies, which cross-reacted with Ole e 1 and Ole e 1-like allergens from ash, lilac and privet pollens. The ability of the human IgE to block the binding of anti-Ole e 1 mutant-specific mouse IgG1 antibodies to natural Ole e 1 demonstrated that Ole e 1 mutants are able to induce in vivo antibodies reactive to the natural allergen. CONCLUSION: The 135Delta10 mutant with reduced allergenicity, intact T cell reactivity and capacity to induce blocking antibodies could provide a suitable candidate vaccine for efficient and safer therapy of olive pollen allergy.

Prophylactic intranasal treatment with fragments of 1,3-beta-glucanase olive pollen allergen prevents airway inflammation in a murine model of type I allergy.

BACKGROUND: Olive pollen is an important cause of allergy in Mediterranean countries. More than 50% of olive-pollen-allergic patients are sensitized against the 1,3-beta-glucanase Ole e 9. To date, prophylactic and therapeutic treatments using purified recombinant allergens have not been studied in animal models of olive pollen allergy. METHODS: BALB/c mice were immunized against Ole e 9 combining intraperitoneal injections of the allergen in Al(OH)3 with airway allergen challenges. A prophylactic treatment was performed by intranasal administration of a mixture of the recombinant fragments of the allergen prior to Ole e 9 sensitization. Serum levels of specific IgE, IgG1, IgG2a and IgG2b were measured by ELISA, and total IgE levels by sandwich ELISA. Bronchoalveolar lavage and lungs from mice were collected to study airway inflammation by light microscopy. RESULTS: BALB/c mice immunized against Ole e 9 developed a predominantly Th2-like immune response with allergen-specific immunoglobulin induction and airway inflammation accompanied by the infiltration of eosinophils, lymphocytes, and neutrophils in the lung. Prophylactic treatment by intranasal application of the recombinant fragments of Ole e 9 avoids airway inflammation induced by sensitization with this allergen although the levels of Ole e 9-specific antibodies remain unchanged. CONCLUSIONS: Prophylactic intranasal treatment with recombinant fragments of Ole e 9 prevents airway inflammation triggered by immunization to this allergen in a murine model of type I allergy.

Isolation and characterization of an olive allergen-like protein from lilac pollen. Sequence analysis of three cDNA encoding protein isoforms.

An olive allergen-like protein has been isolated from lilac (Syringa vulgaris) pollen extract. The protein can be considered as an allergen since is recognized by IgE from olive hypersensitive human sera, and has been called Syr v I (IUIS nomenclature). This protein consists of a glycosylated polypeptide of 20 kDa, which has an amino acid composition, spectroscopic properties, and an N-terminal sequence similar to the major allergen from olive pollen, Ole e I. The lilac allergen is recognized by rabbit polyclonal antisera raised against olive allergen as well as by an Ole e I-specific monoclonal antibody. Using a polymerase chain reaction strategy, based on the similarities observed between these olive and lilac proteins, three cDNA clones encoding Syr v I have been isolated and sequenced. These clones code for a polymorphic protein of 145 residues with a derived molecular mass of about 16,400Da, which contains a potential N-glycosylation site. Comparison of the deduced amino acid sequences of these Syr v I isoforms to each other revealed identities of 90-97%. Moreover, these sequences showed a high degree of similarity (85.5-89.6% identity) with Ole e I. The structural and immunological characterization of Syr v I justify the cross-reactions observed between olive and lilac pollen extracts. The molecular cloning of Syr v I is relevant for the epitope mapping in Oleaceae allergens, and may contribute to an improvement in the design of reagents for diagnosis and therapy of IgE-dependent allergic reactions.