RESUMO
The production of specific antibodies able to recognize allergens from different sources or block interactions between allergens and antibodies mediating allergic reactions is crucial for developing successful tools for diagnostics and therapeutics. Panallergens are highly conserved proteins present in widely different species, implicated in relevant cross-reactions. The panallergen latex profilin (Hev b 8) has been associated with the latex-food-pollen syndrome. We generated five monoclonal IgGs and one IgE from murine hybridomas against recombinant Hev b 8 and evaluated their interaction with this allergen using ELISA and biolayer interferometry (BLI). Affinity purified mAbs exhibited high binding affinities towards rHev b 8, with KD1 values ranging from 10-10 M to 10-11 M. Some of these antibodies also recognized the recombinant profilins from maize and tomato (Zea m 12 and Sola l 1), and the ash tree pollen (Fra e 2). Competition ELISA demonstrated that some mAb pairs could bind simultaneously to rHev b 8. Using BLI, we detected competitive, non-competitive, and partial-competition interactions between pairs of mAbs with rHev b 8, suggesting the existence of at least two non-overlapping epitopes on the surface of this allergen. Three-dimensional models of the Fv of 1B4 and 2D10 IgGs and docking simulations of these Fvs with rHev b 8 revealed these epitopes. Furthermore, these two mAbs inhibited the interaction of polyclonal IgE and IgG4 antibodies from profilin-allergic patients with rHev b 8, indicating that the mAbs and the antibodies present in sera from allergic patients bind to overlapping epitopes on the allergen. These mAbs can be useful tools for immune-localization studies, immunoassay development, or standardization of allergenic products.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Plantas/imunologia , Reações Cruzadas/imunologia , Epitopos/imunologia , Látex/imunologia , Profilinas/imunologia , Alérgenos/imunologia , Sequência de Aminoácidos , Animais , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Hipersensibilidade ao Látex/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Plantas/imunologia , Pólen/imunologiaRESUMO
BACKGROUND: Latex allergy is a public health issue. It presents elevated prevalence in known risk groups, especially in those patients with spine bifida condition, urinal malformation and for those with orthopedic problems - multiple surgeries. Health Services in Mexico do not have the enough studies about prevalence and risk associated to latex allergy. OBJECTIVE: Determine latex sensitization prevalence through PRICK test in patients with genitourinary malformations and more than 3 surgeries in UMAE pediatric CMNO unit, considering too related factors. MATERIAL AND METHOD: An analytical and descriptive cross-sectional study was performed, which included men and women from 1 to 16 years with genitourinary malformations and more than three surgeries performed. A survey to know the risk factors associated and skin puncture test was performed with latex extract, with positive and negative control. Serum levels of total IgE and eosinophils were measured in peripheral blood. RESULTS: The study exposed prevalence of 30.72%. Related to associated factors as follows: atopy (p=0.047), previous antecedent reaction to latex products (p=0.003) specific for balloons (p=0.000) and gloves (p=0.002). There was not association related to amount of surgeries and surgical interventions on early age, either for high levels of total serum IgE. CONCLUSION: Prevalence to latex sensitization is high in risk groups. Especially in those with atopy thereby is important for health personal to identify these patients in order to implement on time the preventive primary/secondary measures. With these actions potential mortal risks like anaphylaxis will be avoided. This will decrease sanitary costs and mortality.
Antecedentes: la alergia al látex tiene alta prevalencia en grupos de riesgo conocidos, especialmente en pacientes con espina bífida, malformaciones urinarias y ortopédicas con múltiples cirugías. En México no se cuenta con suficientes estudios que reporten la prevalencia y factores de riesgo asociados. Objetivo: determinar la prevalencia de sensibilización al látex mediante prueba de prick con extracto de látex en pacientes con malformaciones genitourinarias y más de tres cirugías en la Unidad Médica de Alta Especialidad de Pediatría del Centro Médico Nacional de Occidente, así como los factores asociados. Material y método: Se realizó un estudio transversal analítico y descriptivo, que incluyó hombres y mujeres de 1 a 16 años, con malformaciones genitourinarias y más de tres cirugías. Se aplicó una encuesta para conocer los factores de riesgo asociados y se realizó prueba cutánea por punción con extracto de látex, con control positivo y negativo. Se midieron niveles séricos de IgE total y eosinófilos en sangre periférica. Resultados: la prevalencia encontrada fue de 30.7% y los factores de riesgo asociados: atopia personal (p=0.047), antecedente de reacción previa con productos con látex (p=0.003), específicamente con globos (p=0.000) y guantes (p=0.002). No hubo asociación entre el número de cirugías e intervenciones quirúrgicas a edades tempranas, tampoco con concentraciones elevadas de IgE sérica total. Tuvieron reacción cruzada a frutas-látex 25% de los pacientes, los alimentos asociados fueron: aguacate, papaya, fresa y kiwi. Conclusión: la prevalencia de sensibilización al látex es alta en los grupos de riesgo, sobre todo con antecedente de atopia, por lo que es importante que el personal de salud identifique a estos pacientes a fin de implementar oportunamente las medidas de prevención primaria y secundaria; evitar efectos severos potencialmente mortales, como la anafilaxia, para disminuir la morbilidad y la mortalidad y los costos sanitarios.
Assuntos
Hipersensibilidade ao Látex/epidemiologia , Anormalidades Urogenitais/imunologia , Adolescente , Criança , Pré-Escolar , Estudos Transversais , Eosinófilos/citologia , Feminino , Humanos , Imunoglobulina E/sangue , Lactente , Látex/imunologia , Hipersensibilidade ao Látex/imunologia , Masculino , México/epidemiologia , Prevalência , Fatores de Risco , Testes Cutâneos/estatística & dados numéricos , Anormalidades Urogenitais/cirurgiaRESUMO
Latex allergy is a health problem that mainly affects medical environments, causing anaphylactic shocks in extreme cases. Sensitization and reactions to this material is closely linked to the use of latex gloves. The objective of this study was to purify two of the major allergens from latex surgical gloves to study the biochemical and structural changes that could be generated during the product manufacture and to compare their IgE recognition with the non-processed allergens. Glycosylated allergen Hev b 2 (ß-1,3-glucanase) and Hev b 6.02 (hevein) were purified from glove extracts using affinity (Concanavalin A) and reversed-phase chromatographies, respectively. ELISA experiments were performed with both proteins and sera from allergic patients to assess the IgE recognition, which was heterogeneous. Crystallographic methods were used to obtain the 3D structure of Hev b 6.02 from surgical gloves, which did not show evident modification when compared with the protein from the natural non-processed form. Despite having the same crystallographic structure, the IgE from some patients showed different recognition when the glove and the natural allergen were used in ELISA. Furthermore, using electrophoretic techniques, we identified three forms of Hev b 2: one corresponding to the complete polypeptide chain with posttranslational modifications, and two glycosylated fragments. The mixture of these three forms showed stronger recognition by IgE from latex-allergic patients than the pure non-processed allergen. In conclusion, IgE from subjects sensitized to latex products showed different recognition between the allergens obtained from a natural source and the processed material, even when the structure was maintained. This demonstrates the importance of using processed allergens in further investigations of diagnosis, prevalence, product allergenicity, and therapies.
Assuntos
Antígenos de Plantas , Peptídeos Catiônicos Antimicrobianos , Luvas Cirúrgicas/efeitos adversos , Imunoglobulina E , Hipersensibilidade ao Látex/imunologia , Látex , Lectinas de Plantas , Proteínas de Plantas , Antígenos de Plantas/química , Antígenos de Plantas/imunologia , Antígenos de Plantas/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/imunologia , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Cristalografia por Raios X , Feminino , Hevea/química , Humanos , Imunoglobulina E/química , Imunoglobulina E/imunologia , Látex/efeitos adversos , Látex/química , Masculino , Lectinas de Plantas/química , Lectinas de Plantas/imunologia , Lectinas de Plantas/isolamento & purificação , Proteínas de Plantas/química , Proteínas de Plantas/imunologia , Proteínas de Plantas/isolamento & purificação , Processamento de Proteína Pós-Traducional/imunologia , Relação Estrutura-AtividadeAssuntos
Antígenos de Plantas/imunologia , Basófilos/imunologia , Hevea/imunologia , Hipersensibilidade ao Látex/diagnóstico , Hipersensibilidade ao Látex/imunologia , Proteínas de Plantas/imunologia , Adulto , Antígenos de Plantas/química , Antígenos de Plantas/farmacologia , Basófilos/efeitos dos fármacos , Basófilos/patologia , Células Cultivadas , Criança , Feminino , Expressão Gênica , Hevea/química , Humanos , Imunoglobulina E/sangue , Hipersensibilidade ao Látex/genética , Hipersensibilidade ao Látex/patologia , Masculino , Pessoa de Meia-Idade , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/imunologia , Proteínas de Plantas/química , Proteínas de Plantas/farmacologia , Pirofosfatases/genética , Pirofosfatases/imunologia , Testes Cutâneos , Tetraspanina 30/genética , Tetraspanina 30/imunologiaRESUMO
Endogenous glycosylated Hev b 2 (endo-ß-1,3-glucanase) from Hevea brasiliensis is an important latex allergen that is recognized by IgE antibodies from patients who suffer from latex allergy. The carbohydrate moieties of Hev b 2 constitute a potentially important IgE-binding epitope that could be responsible for its cross-reactivity. Here, the structure of the endogenous isoform II of Hev b 2 that exhibits three post-translational modifications, including an N-terminal pyroglutamate and two glycosylation sites at Asn27 and at Asn314, is reported from two crystal polymorphs. These modifications form a patch on the surface of the molecule that is proposed to be one of the binding sites for IgE. A structure is also proposed for the most important N-glycan present in this protein as determined by digestion with specific enzymes. To analyze the role of the carbohydrate moieties in IgE antibody binding and in human basophil activation, the glycoallergen was enzymatically deglycosylated and evaluated. Time-lapse automated video microscopy of basophils stimulated with glycosylated Hev b 2 revealed basophil activation and degranulation. Immunological studies suggested that carbohydrates on Hev b 2 represent an allergenic IgE epitope. In addition, a dimer was found in each asymmetric unit that may reflect a regulatory mechanism of this plant defence protein.
Assuntos
Antígenos de Plantas/química , Basófilos/efeitos dos fármacos , Celulase/química , Hevea/química , Imunoglobulina E/química , Proteínas de Plantas/química , Sequência de Aminoácidos , Antígenos de Plantas/imunologia , Antígenos de Plantas/isolamento & purificação , Antígenos de Plantas/farmacologia , Teste de Degranulação de Basófilos , Basófilos/citologia , Basófilos/imunologia , Sítios de Ligação , Sequência de Carboidratos , Células Cultivadas , Celulase/imunologia , Celulase/isolamento & purificação , Celulase/farmacologia , Cristalografia por Raios X , Ácido Glutâmico/metabolismo , Glicosilação , Humanos , Imunoglobulina E/imunologia , Hipersensibilidade ao Látex/imunologia , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Plantas/imunologia , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Ligação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Quaternária de Proteína , Imagem com Lapso de TempoRESUMO
Natural rubber latex (NRL; Hevea brasiliensis) allergy is an IgE-mediated reaction to latex proteins. When latex glove exposure is the main sensitizing agent, Hev b 5 is one of the major allergens. Dendritic cells (DC), the main antigen presenting cells, modulated with pharmacological agents can restore tolerance in several experimental models, including allergy. In the current study, we aimed to generate DC with tolerogenic properties from NRL-allergic patients and evaluate their ability to modulate allergen-specific T and B cell responses. Here we show that dexamethasone-treated DC (dxDC) differentiated into a subset of DC, characterized by low expression of MHC class II, CD40, CD80, CD86 and CD83 molecules. Compared with LPS-matured DC, dxDC secreted lower IL-12 and higher IL-10 after CD40L activation, and induced lower alloantigenic T cell proliferation. We also show that dxDC pulsed with the dominant Hev b 5 T-cell epitope peptide, Hev b 5(46-65), inhibited both proliferation of Hev b 5-specific T-cell lines and the production of Hev b 5-specific IgE. Additionally, dxDC induced a subpopulation of IL-10-producing regulatory T cells that suppressed proliferation of Hev b 5-primed T cells. In conclusion, dxDC generated from NRL-allergic patients can modulate allergen-specific T-cell responses and IgE production, supporting their potential use in allergen-specific immunotherapy.
Assuntos
Alérgenos/imunologia , Células Dendríticas/imunologia , Imunoglobulina E/imunologia , Hipersensibilidade ao Látex/imunologia , Linfócitos T/imunologia , Adulto , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos de Plantas/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Linhagem Celular , Proliferação de Células , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Dexametasona/farmacologia , Epitopos de Linfócito T/imunologia , Feminino , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Interleucina-10/imunologia , Interleucina-10/metabolismo , Masculino , Pessoa de Meia-Idade , Peptídeos/imunologia , Proteínas de Plantas/imunologia , Linfócitos T/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Adulto JovemRESUMO
BACKGROUND: Latex allergy is an important cause of occupational allergy. In many countries the prevalence of latex allergy is still high and the profile of latex sensitization is unknown. OBJECTIVES: To evaluate the frequency of sensitization and allergy to latex in children and adolescents with myelomeningocele and to identify associated risk factors. METHODS: The study included 55 children and adolescents with myelomeningocele followed at a specialized center. In addition to a standard questionnaire and skin prick tests (SPTs) to aeroallergens and total latex, the patients underwent determination of total and specific serum IgE to latex and recombinant allergens. RESULTS: We observed a prevalence of 25% for latex sensitization and of 20% for latex allergy. Twenty-four patients (43.6%) were atopic and the average age for the first reaction to latex was 44.5 months. Cutaneous reactions were the most frequently reported reactions (72.7%). Specific immunoglobulin (Ig) E to rHev b 1, rHev b 3, rHev b 5, rHev b 6.1, and rHev b 6.2 was detected in over 50% of patients allergic to latex. Multivariate analysis showed current asthma, atopy, and having undergone 4 or more operations to be risk factors for latex sensitization. CONCLUSIONS: Our study documented a high prevalence of sensitization and allergy to latex in patients with myelomeningocele. Specific IgE to rHev b 1, rHev b 3, rHev b 5, rHev b 6.1, and rHev b 6.2 was detected in over 50% of children and adolescents with myelomeningocele who are allergic to latex. A history of current asthma, atopy, and having undergone 4 or more operations were independent risk factors for latex allergy.
Assuntos
Alérgenos/imunologia , Hipersensibilidade ao Látex/complicações , Hipersensibilidade ao Látex/epidemiologia , Látex/imunologia , Meningomielocele/complicações , Meningomielocele/epidemiologia , Adolescente , Brasil/epidemiologia , Criança , Feminino , Humanos , Hipersensibilidade ao Látex/imunologia , Masculino , Prevalência , Fatores de RiscoRESUMO
SCOPE: Manioc (Manihot esculenta) is a tuber mainly consumed in the Southern Hemisphere and used worldwide by food and chemistry industry. We aimed to recombinantly produce and characterize the first manioc allergen and evaluate its IgE reactivity in sera of Brazilian and Italian patients. METHODS AND RESULTS: The molecule, termed Man e5, was expressed in E. coli, characterized by amino acid analysis, mass spectrometry, circular dichroism, HPLC, and dynamic light scattering. A tertiary structural model of the protein was produced using bioinformatics and susceptibility to pepsin digestion was analyzed in vitro. Based on its high content of charged residues, heat stability, flexibility and lack of secondary structure elements, the allergen was determined a member of the intrinsically disordered protein family. Brazilian patients were selected based on manioc allergy and Italians based on latex allergy and sensitization to Hev b 5.71% of Brazilians and 40% of Italians were in vitro IgE positive to Man e5. Cross-inhibition assays suggest a possible involvement of this allergen in the latex-fruit syndrome. CONCLUSION: Man e5, the first purified allergen from manioc demonstrates IgE cross-reactivity with Hev b 5. Data suggest Hev b 5 might act as primary sensitizer and could therefore lead to allergic manifestations upon manioc consumption without prior exposition.
Assuntos
Alérgenos/genética , Antígenos de Plantas/genética , Antígenos de Plantas/imunologia , Hipersensibilidade Alimentar/imunologia , Manihot/química , Proteínas de Plantas/imunologia , Adulto , Alérgenos/imunologia , Sequência de Aminoácidos , Brasil , Dicroísmo Circular , Clonagem Molecular , Reações Cruzadas/imunologia , Feminino , Hipersensibilidade Alimentar/sangue , Hipersensibilidade Alimentar/diagnóstico , Humanos , Soros Imunes , Imunoglobulina E/imunologia , Látex/imunologia , Hipersensibilidade ao Látex/sangue , Hipersensibilidade ao Látex/imunologia , Masculino , Manihot/imunologia , Pessoa de Meia-Idade , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
Diversos estudios han demostrado que entre un 20% y un 60% de los individuos alérgicos a látex presentan reacciones mediadas por inmunoglobulina E (IgE) a una amplia variedad de alimentos, principalmente a frutas como el cambur (banana, plátano) el aguacate, la castaña y el kiwi, lo cual se conoce como síndrome látex-frutas. El odontólogo debe conocer esta reacción cruzada de alergia a fin de evitar situaciones que puedan poner en riesgo al paciente si no se toman las medidas que garanticen un ambiente seguro libre de látex
Studies have shown that between 20% and 60% of individuals allergic to latex have IgE-mediated reactions (IgE) to a wide variety of foods, mainly fruits like banana, avocado, sweet chestnut and kiwi, which is called latex-fruit syndrome. The dentist should be aware of this allergy cross-reaction to avoid situations that may endanger the patient if not taken measures to ensure a safe environment free of latex
Assuntos
Humanos , Masculino , Feminino , Hipersensibilidade Alimentar , Hipersensibilidade ao Látex/diagnóstico , Hipersensibilidade ao Látex/imunologia , Odontologia GeralRESUMO
BACKGROUND: Solidago virgaurea (goldenrod) is a perennial weed from which no allergens have been identified. A high latex content in its leaves has been reported. Although not an airborne allergen, it may be an important occupational sensitizer. OBJECTIVE: To identify allergenic proteins in goldenrod and to determine whether they cross-react with Hevea brasiliensis latex. METHODS: Potential cross-reactive allergens in latex and goldenrod were investigated by immunoblot inhibition and ImmunoCAP inhibition analyses using serum from patients with clinically evident goldenrod and/or latex allergy. Cross reactivity between latex allergens and goldenrod proteins was studied using recombinant Hev b 1, 3, 4, 5, 6.01, 6.02, 8, 9, or 11 in ImmunoCAP inhibition analyses. RESULTS: Immunoglobulin (Ig) E antibodies from individuals with goldenrod allergy bound extracted goldenrod proteins ranging from 20 kDa to 130 kDa in Western blots. Evidence for latex and goldenrod cross reactivity was identified by ImmunoCAP and immunoblot inhibition experiments using serum from patients with strongly positive concomitant latex and goldenrod-specific IgE antibody responses. Observed latex-goldenrod cross reactivity could not be ascribed to any of the recombinant major latex allergens evaluated. CONCLUSIONS: H brasiliensis latex and goldenrod contain cross-reactive and unique allergenic proteins. Exposure to goldenrod may sensitize patients to latex and vice versa.
Assuntos
Antígenos de Plantas/imunologia , Reações Cruzadas/imunologia , Epitopos/imunologia , Hipersensibilidade ao Látex/imunologia , Rinite Alérgica Sazonal/imunologia , Ligação Competitiva , Western Blotting , Feminino , Pessoal de Saúde , Hevea/imunologia , Humanos , Imunoglobulina E/sangue , Hipersensibilidade ao Látex/sangue , Hipersensibilidade ao Látex/diagnóstico , Pessoa de Meia-Idade , Exposição Ocupacional/efeitos adversos , Rinite Alérgica Sazonal/sangue , Rinite Alérgica Sazonal/diagnóstico , Solidago/imunologiaRESUMO
BACKGROUND: Component-resolved diagnosis using microarray technology has recently been introduced in clinical allergology, but its applicability in patients with natural rubber latex (NRL) allergy has not been investigated. OBJECTIVES: To evaluate the utility of microarray-based immunoglobulin (Ig) E detection in the diagnostic workup of NRL allergy and to compare this new diagnostic tool with established methods of NRL-specific IgE detection. METHODS: We investigated 52 adults with immediate-type NRL allergy and 50 control patients. Determination of specific serum IgE against 8 recombinant Hevea brasiliensis allergen components was performed using a customized allergen microarray and a conventional fluorescence enzyme immunoassay (FEIA). RESULTS: The panel of microarrayed allergen components was shown to represent a comprehensive repertoire of clinically relevant NRL proteins. NRL-specific IgE recognition patterns and sensitization rates determined by microarray analysis were similar to those obtained by conventional FEIA. The diagnostic sensitivity rates of combined single-component data were not significantly different for the respective recombinant test system, whereas the sensitivity level of extract-based FEIA analysis was markedly higher. CONCLUSION: The current study provides evidence that microarrays of recombinant NRL allergen components are a suitable new tool for the diagnosis of NRL-specific sensitization.They show performance characteristics comparable to those of current diagnostic tests and could be indicated in small children in whom only limited blood volumes are obtainable. Further large-scale studies in unselected patient populations and in high-risk groups are warranted before the microarray can be introduced into routine management of patients with NRL allergy.
Assuntos
Antígenos de Plantas , Hevea/imunologia , Hipersensibilidade ao Látex/diagnóstico , Análise Serial de Proteínas , Proteínas Recombinantes , Adulto , Idoso , Antígenos de Plantas/imunologia , Progressão da Doença , Epitopos/metabolismo , Feminino , Imunoensaio de Fluorescência por Polarização , Humanos , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Hipersensibilidade ao Látex/imunologia , Hipersensibilidade ao Látex/fisiopatologia , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes/imunologiaRESUMO
The proteome of Hevea brasiliensis latex has been explored in depth via combinatorial peptide ligand libraries. A total of 300 unique gene products have been identified in this latex, whose proteome has been largely unknown up to the present. In search for unknown allergens, control latex and eluates from the ligand libraries have been fractionated by two-dimensional mapping, blotted and confronted with sera of 18 patients. In addition to the already known and named Hevea major allergens, we have unambiguously detected several others like, for instance: heat shock protein (81 kDa), proteasome subunit (30 kDa), protease inhibitor (8 kDa), hevamine A (43 kDa) and glyceraldehyde-3-phosphate dehydrogenase (37 kDa). Gene Ontology analysis of analyzed fractions has shown that major functions are substantially unchanged after sample treatment, while novel biological functions appeared that were undetectable in the crude sample.
Assuntos
Antígenos de Plantas/química , Antígenos de Plantas/imunologia , Hevea/imunologia , Látex/imunologia , Biblioteca de Peptídeos , Proteoma/química , Proteoma/imunologia , Técnicas de Química Combinatória/métodos , Humanos , Hipersensibilidade ao Látex/imunologiaRESUMO
Hev b 6.02 (hevein), identified as a major allergen from natural rubber latex (NRL), is involved in the latex-fruit syndrome and also acts as a pathogenesis defense-related protein. Its 3D structure has been solved at high resolution, and its linear epitopes have already been reported. However, information about conformational epitopes is still controversial, even though it is relevant for an accurate diagnosis and treatment, as well as for the study of allergen-antibody molecular interactions. We sought to analyze the B-cell epitopes of Hev b 6.02 at a molecular and structural level, using specific recombinant antibodies. We obtained a murine monoclonal antibody (mAb 6E7) and three human single chain fragments (scFvs A6, H8, and G7) anti-Hev b 6.02 that were able to compete for hevein binding with serum IgEs from latex allergic patients. In vitro assays showed that the mAb 6E7 and scFv H8 recognized the area of Hev b 6.02 where the aromatic residues are exposed; while the scFv G7 defined the amino and carboxy-terminal regions that lie close to each other, as a different epitope. The structural modeling of the Hev b 6.02-scFv H8 and Hev b 6.02-scFv G7 complexes revealed the putative regions of two conformational epitopes. In one of these, the aromatic residues, as well as polar side chains are important for the interaction, suggesting that they are part of a dominant conformational epitope also presented on the Hev b 6.02-IgE interactions. Antibodies recognizing this important allergen have potential to be used to diagnose and ultimately treat latex allergy.
Assuntos
Alérgenos/química , Peptídeos Catiônicos Antimicrobianos/química , Mapeamento de Epitopos , Epitopos de Linfócito B/química , Hipersensibilidade ao Látex/imunologia , Lectinas de Plantas/química , Alérgenos/imunologia , Sequência de Aminoácidos , Anticorpos Bloqueadores/imunologia , Anticorpos Monoclonais/imunologia , Peptídeos Catiônicos Antimicrobianos/imunologia , Epitopos de Linfócito B/imunologia , Humanos , Imunoglobulina E/sangue , Dados de Sequência Molecular , Lectinas de Plantas/imunologia , Conformação Proteica , Alinhamento de SequênciaRESUMO
Several proteins of rubber latex have been recognized as allergens causing immediate hypersensitivity in humans. In this study, a bottom fraction membrane (BFM) protein preparation from Hevea brasiliensis trees grown in southern Thailand was used to detect specific IgE in four groups of serum samples. The first group included 170 samples of latex glove factory workers (LGWs); group 2 consisted of the sera of 35 health care workers (HCWs) who were repeatedly exposed to powdered latex gloves; groups 3 and 4 were 31 positive and 22 negative sera, respectively, obtained from Johns Hopkins University School of Medicine, Baltimore, USA, tested for IgE to latex allergen. It was found that 56/170 (33%), 5/35 (14%), 11/31 (35.5%) and 1/22 (4.5%) samples of the LGWs, HCWs, CAP+ and CAP- groups had significant IgE to the BFM proteins, respectively. However, of all subjects only one subject of group 1 had experienced allergic morbidity consisting of eczema, conjunctivitis and asthma. The IgE of this subject bound to a 55 kDa component in the rubber latex BFM preparation. Thus, this protein may be regarded as a novel, although minor, latex allergen. Further investigation is needed to characterize the component and to pinpoint its allergenic role.
Assuntos
Alérgenos/imunologia , Hevea , Hipersensibilidade ao Látex/imunologia , Proteínas de Plantas/imunologia , Alérgenos/isolamento & purificação , Fracionamento Celular , Luvas Protetoras/efeitos adversos , Setor de Assistência à Saúde , Pessoal de Saúde , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imunoglobulina E/sangue , Hipersensibilidade ao Látex/sangue , Membranas , Exposição Ocupacional , Proteínas de Plantas/isolamento & purificação , Borracha/efeitos adversosRESUMO
Pt2L4 is a protein from cassava homologue to Hevb5, a principal allergen from latex. Here we aimed to elucidate immunological relationships between these proteins. Our results revealed that epitopes found in Hev b 5 are not entirely conserved in Pt2L4 which is not recognized by IgE from patients allergic to Hev b 5.
Assuntos
Alérgenos/imunologia , Hipersensibilidade ao Látex/imunologia , Manihot/química , Proteínas de Plantas/imunologia , Alérgenos/química , Sequência de Aminoácidos , Antígenos de Plantas , Western Blotting , Reações Cruzadas , Epitopos/imunologia , Ácido Glutâmico , Hevea/química , Hevea/imunologia , Humanos , Imunoglobulina G/imunologia , Látex/imunologia , Manihot/imunologia , Dados de Sequência Molecular , Proteínas de Plantas/química , Raízes de Plantas/química , Raízes de Plantas/genética , Raízes de Plantas/imunologia , Homologia Estrutural de ProteínaRESUMO
BACKGROUND: Characterized native and recombinant Hevea brasiliensis (rHev b) natural rubber latex (NRL) allergens are available to assess patient allergen sensitization profiles. OBJECTIVE: Quantification of individual IgE responses to the spectrum of documented NRL allergens and evaluation of cross-reactive carbohydrate determinants (CCDs) for more definitive diagnosis. METHODS: Sera of 104 healthcare workers (HCW; 51 German, 21 Portuguese, 32 American), 31 spina bifida patients (SB; 11 German, 20 Portuguese) and 10 Portuguese with multiple surgeries (MS) were analysed for allergen-specific IgE antibody (sIgE) to NRL, single Hev b allergens and CCDs with ImmunoCAP technology. RESULTS: In all patient groups rHev b 5-sIgE concentrations were the most pronounced. Hev b 2, 5, 6.01 and 13 were identified as the major allergens in HCW and combined with Hev b 1 and Hev b 3 in SB. In MS Hev b 1 displayed an intermediate relevance. Different sIgE antibody levels to native Hevea brasiliensis (nHev b) 2 and rHev b 6.01 allowed discrimination of SB with clinical relevant latex allergy vs. those with latex sensitization. Sensitization profiles of German, Portuguese and American patients were equivalent. rHev b 5, 6.01 and nHev b 13 combined detected 100% of the latex-allergic HCW and 80.1% of the SB. Only 8.3% of the sera showed sIgE response to CCDs. CONCLUSIONS: Hev b 1, 2, 5, 6.01 and 13 were identified as the major Hev b allergens and they should be present in standardized latex extracts and in vitro allergosorbents. CCDs are only of minor relevance in patients with clinical relevant latex allergy. Component-resolved diagnostic analyses for latex allergy set the stage for an allergen-directed immunotherapy strategy.
Assuntos
Reações Antígeno-Anticorpo/imunologia , Imunoglobulina E/imunologia , Hipersensibilidade ao Látex/diagnóstico , Borracha , Adolescente , Adulto , Antígenos de Plantas/biossíntese , Antígenos de Plantas/genética , Antígenos de Plantas/imunologia , Carboidratos/imunologia , Criança , Pré-Escolar , Reações Cruzadas/imunologia , Epitopos/imunologia , Feminino , Alemanha , Pessoal de Saúde , Hevea/química , Hevea/genética , Humanos , Imunoglobulina E/sangue , Hipersensibilidade ao Látex/sangue , Hipersensibilidade ao Látex/imunologia , Masculino , Pessoa de Meia-Idade , Portugal , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Disrafismo Espinal/complicações , Estados UnidosRESUMO
BACKGROUND: The latex of Hevea brasiliensis trees contains a complex proteome that includes a range of allergenic proteins. Current latex extracts that are used for the diagnosis of latex allergy still lack important allergens. We aimed to devise a production process for an improved reagent that would ideally contain the complete latex allergome. METHODS: Latex C-serum was fractionated by ammonium sulfate precipitation, and B- and C-serum proteins were then separated by anion exchange chromatography. Proteins eluting within defined salt concentration ranges were pooled into six final fractions. Fractions were evaluated by two-dimensional electrophoresis and subsequent IgE immunoblot for their spectrum of allergens. The presence of the most important latex allergens in the fractions was checked by Western blot analyses. Each fraction was further evaluated by skin prick test (SPT). RESULTS: Reproducibility of the preparation method was demonstrated with two batches of latex. Comparison of latex B- and C-serum to the six fractions showed a remarkable increase in the number of detectable allergens in the fractions. The presence of the latex allergens Hev b 1-8 and Hev b 13 in the fractions was demonstrated. In SPTs, the fractions produced wheal-and-flare reactions comparable to commercial latex extracts. CONCLUSIONS: This method provides reproducible latex protein fractions of high allergen content for the diagnosis of latex allergy.
Assuntos
Alérgenos/metabolismo , Antígenos de Plantas/metabolismo , Hevea/imunologia , Hipersensibilidade ao Látex/diagnóstico , Látex/imunologia , Látex/metabolismo , Proteínas de Plantas/metabolismo , Alérgenos/imunologia , Antígenos de Plantas/imunologia , Antígenos de Plantas/isolamento & purificação , Humanos , Látex/isolamento & purificação , Hipersensibilidade ao Látex/imunologia , Proteínas de Plantas/imunologia , Testes CutâneosRESUMO
Immunological and allergenic responses against the latex of Calotropis procera were investigated in mice by oral and subcutaneous routes. The latex was fractionated according to water solubility and molecular size of its components. The fractions were named as non-dialyzable latex (NDL) corresponding to the major latex proteins, dialyzable latex (DL) corresponding to low molecular size substances and rubber latex (RL) which was highly insoluble in water. Anti-sera against these fractions were assayed for total IgG and IgA titration by ELISA and IgE and IgG(1) were quantified by passive cutaneous anaphylaxis (PCA) in rats and mice, respectively. None of the fractions induced antibodies level increases when mice received latex fractions by oral route and thus, did not develop allergy. Nonetheless, anti-sera of mice sensitized with NDL and RL by subcutaneous route displayed considerable immunological response while DL did not. IgG level augmented consistently against NDL and RL while IgA response was detected only to NDL. NDL and RL induced very strong PCA reactions suggesting that both fractions would contain latex substances involved in allergy. Furthermore, protein analysis of NDL and RL suggests that RL still retain residual proteins abundantly found in NDL that could explain its similar allergenic effect. No IgG(1) reaction was detected in any of the anti-sera tested. According to the results, the proteins of latex of Calotropis procera can provoke allergy by subcutaneous route. The NDL has previously shown to display anti-inflammatory and analgesic activities by intraperitoneal injection. It should be relevant to determine whether NDL could induce such activities when assayed by oral route since it was ineffective to induce allergy by this way.
Assuntos
Formação de Anticorpos , Antígenos de Plantas/administração & dosagem , Antígenos de Plantas/farmacologia , Calotropis , Hipersensibilidade ao Látex/imunologia , Látex/administração & dosagem , Látex/imunologia , Administração Oral , Animais , Antígenos de Plantas/química , Brasil , Fracionamento Químico , Relação Dose-Resposta Imunológica , Ensaio de Imunoadsorção Enzimática , Imunização , Imunoglobulina A/sangue , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Injeções Subcutâneas , Látex/química , Masculino , Camundongos , Peso Molecular , Anafilaxia Cutânea Passiva , Extratos Vegetais/administração & dosagem , Extratos Vegetais/imunologia , Ratos , Solubilidade , Solventes/química , Fatores de Tempo , Água/químicaRESUMO
BACKGROUND: Lipid transfer proteins (LTPs) are relevant allergens in certain plants. The role of the LTP of Hevea brasiliensis in the latex-fruit syndrome is widely unknown. OBJECTIVE: To study IgE reactivity with recombinant Hevea LTP in sera of fruit-allergic adults with and without natural rubber latex (NRL) allergy. METHODS: An LTP-specific complementary DNA of H brasiliensis leaves was amplified, subcloned into the pMAL expression system, and analyzed. The recombinant protein was coupled to ImmunoCAP, and the IgE-binding properties were studied in sera of 10 NRL-allergic patients without symptoms to fruit and 48 atopic patients with fruit allergy. Eleven of these 48 patients were also allergic to NRL, 14 displayed sensitization to NRL without symptoms on NRL exposure so far, and 23 had neither symptoms nor IgE antibodies to NRL. RESULTS: After expression in Escherichia coli, a soluble maltose-binding protein-rHev b 12 fusion protein was isolated and coupled to ImmunoCAP to determine rHev b 12 specific IgE reactivity. rHev b 12 specific IgE binding was found in 3 fruit-allergic patients with NRL sensitization (0.68, 0.88, and 0.96 kU/L) and in 3 fruit-allergic patients without NRL sensitization (1.58, 2.25, and 2.27 kU/L). The remaining 52 serum samples and all maltose-binding protein control test results were negative (< 0.35 kU/L). CONCLUSIONS: In these patients, rHev b 12 specific IgE reactivity seems to result from common cross-reactive epitopes with some of the fruit LTPs tested and underscores only an involvement in co-recognition. No clinical relevance of IgE binding to the LTP of H brasiliensis in association with NRL allergy was detected.