RESUMO
BACKGROUND: Yellow jacket hyaluronidase (YJ-HYA) is considered a major allergen in yellow jacket allergy. It shows 50% homology with the hyaluronidase from honeybee venom, Api m 2. Recently, IgE binding to YJ-HYA and cross-reactivity with Api m 2 has been shown to be due to cross-reactive carbohydrate determinants (CCDs). OBJECTIVE: We sought to quantify the importance of YJ-HYA in yellow jacket allergy and the cross-reactivity with Api m 2 by discriminating between carbohydrate and peptide epitopes. METHODS: IgE binding to Vespula species venom was studied by means of Western blotting in 136 patients with yellow jacket allergy (31 in vitro single positive to yellow jacket venom and 105 in vitro double-positive to yellow jacket-honeybee). Inhibition studies were carried out with MUXF-BSA (isolated bromelain glycopeptides linked to bovine serum albumin) and purified Api m 2. RESULTS: Among yellow jacket single-positive sera, only 1 of 31 bound with YJ-HYA, whereas this was the case in 87% of 105 double-positive sera. Of 83 patients in whom inhibitions were performed, 65% reacted with hyaluronidase through CCDs alone, 27% reacted with both CCDs and peptide epitopes, and 8% reacted only with the hyaluronidase peptide. The protein-specific reactivity with YJ-HYA was cross-inhibited by Api m 2 in 48% (14/29). Antigen 5 and phospholipase A(1) were each recognized by around 90% of sera from both groups, together identifying 97% of patients. CONCLUSION: Hyaluronidase is a minor yellow jacket venom allergen, and only 10% to 15% of patients with yellow jacket allergy are estimated to have IgE against the hyaluronidase protein. Peptide-specific cross-reactivity with Api m 2 occurs in half of these sera. Component-resolved diagnosis with antigen 5 and phospholipase would detect virtually all patients with yellow jacket venom allergy.
Assuntos
Alérgenos , Hialuronoglucosaminidase , Hipersensibilidade Imediata , Mordeduras e Picadas de Insetos/imunologia , Venenos de Vespas/enzimologia , Vespas , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Alérgenos/efeitos adversos , Alérgenos/imunologia , Animais , Antígenos de Plantas , Abelhas/imunologia , Criança , Reações Cruzadas , Feminino , Humanos , Hialuronoglucosaminidase/efeitos adversos , Hialuronoglucosaminidase/imunologia , Hipersensibilidade Imediata/etiologia , Hipersensibilidade Imediata/imunologia , Imunoglobulina E/sangue , Masculino , Pessoa de Meia-Idade , Venenos de Vespas/efeitos adversos , Venenos de Vespas/imunologia , Adulto JovemRESUMO
BACKGROUND: Atopic dermatitis has been thought to be associated with a disturbance in n-6 polyunsaturated fatty acid (PUFA) metabolism, but randomized trials investigating the clinical efficacy of oral supplementation with gammalinolenic acid have revealed conflicting results. AIM OF THE STUDY: To re-investigate the proposed linkage between PUFA dysregulation and atopic dermatitis. MATERIALS AND METHODS: Plasma levels of linoleic acid (LA), gammalinolenic acid (GLA), dihomogammalinolenic acid (DGLA) and arachidonic acid (AA) were measured using HPLC in 22 children with atopic dermatitis. Patients were subdivided into those with elevated total serum IgE (group A, n = 15, IgE > +1 SD of age-specific normal values) and those with normal IgE (group B, n = 7) and compared with children suffering from allergic rhinitis/asthma (group C, n = 8) and with non-atopic controls (group D, n = 6). RESULTS: GLA levels were significantly lower (p < 0.05) in eczema patients with elevated IgE (A, 0.19 +/- 0.06%) and in atopic controls (C, 0.23 +/- 0.06%) than in eczema patients with low IgE (B, 0.42 +/- 0.19%) and non-atopic controls (D, 0.43 +/- 0.16%). There were no significant differences between groups for LIN, DGLA and AA, except for lower LIN levels in atopic controls. Correlation of individual LA and GLA values showed significantly steeper regression lines in low-IgE responders (B and D, k(x) = 0.058) than in high-IgE responders (A and C, k(x) = 0.012; p < 0.02), suggesting impaired delta-6-desaturase function in the latter. For the study population as a whole, there was a clear negative correlation between total levels of IgE and GLA (r(s) = -0.64) and a moderate negative correlation between total IgE and AA (r(s) = -0.38). CONCLUSIONS: Dysregulation of n-6 PUFA metabolism is neither consistently found in nor specifically associated with atopic dermatitis but rather appears to be associated with IgE production and atopy in general. The finding of decreased GLA levels in eczema patients with elevated total IgE and in children with allergic rhinitis and asthma but not in eczema patients with normal total IgE questions the proposed pathophysiologic role of fatty acid dysregulation in atopic dermatitis.
Assuntos
Dermatite Atópica/sangue , Ácidos Graxos Insaturados/sangue , Imunoglobulina E/sangue , Medição de Risco/métodos , Asma/sangue , Asma/diagnóstico , Asma/epidemiologia , Biomarcadores/sangue , Criança , Dermatite Atópica/diagnóstico , Dermatite Atópica/epidemiologia , Feminino , Humanos , Masculino , Prevalência , Prognóstico , Rinite Alérgica Perene/sangue , Rinite Alérgica Perene/diagnóstico , Rinite Alérgica Perene/epidemiologia , Fatores de RiscoRESUMO
BACKGROUND: Double sensitization (DS) to bee and vespid venom is frequently observed in the diagnosis of hymenoptera venom allergy, but clinically relevant DS is rare. Therefore it is sophisticated to choose the relevant venom for specific immunotherapy and overtreatment with both venoms may occur. We aimed to compare currently available routine diagnostic tests as well as experimental tests to identify the most accurate diagnostic tool. METHODS: 117 patients with a history of a bee or vespid allergy were included in the study. Initially, IgE determination by the ImmunoCAP, by the Immulite, and by the ADVIA Centaur, as well as the intradermal test (IDT) and the basophil activation test (BAT) were performed. In 72 CAP double positive patients, individual IgE patterns were determined by western blot inhibition and component resolved diagnosis (CRD) with rApi m 1, nVes v 1, and nVes v 5. RESULTS: Among 117 patients, DS was observed in 63.7% by the Immulite, in 61.5% by the CAP, in 47.9% by the IDT, in 20.5% by the ADVIA, and in 17.1% by the BAT. In CAP double positive patients, western blot inhibition revealed CCD-based DS in 50.8%, and the CRD showed 41.7% of patients with true DS. Generally, agreement between the tests was only fair and inconsistent results were common. CONCLUSION: BAT, CRD, and ADVIA showed a low rate of DS. However, the rate of DS is higher than expected by personal history, indicating that the matter of clinical relevance is still not solved even by novel tests. Furthermore, the lack of agreement between these tests makes it difficult to distinguish between bee and vespid venom allergy. At present, no routinely employed test can be regarded as gold standard to find the clinically relevant sensitization.
Assuntos
Venenos de Abelha/toxicidade , Hipersensibilidade/diagnóstico , Venenos de Vespas/toxicidade , Adulto , Diagnóstico Diferencial , Feminino , Humanos , Hipersensibilidade/etiologia , Masculino , Pessoa de Meia-IdadeRESUMO
On the basis of IgE epitope mapping data, we have produced three allergen fragments comprising aa 1-33, 1-57, and 31-110 of the major timothy grass pollen allergen Phl p 6 aa 1-110 by expression in Escherichia coli and chemical synthesis. Circular dichroism analysis showed that the purified fragments lack the typical alpha-helical fold of the complete allergen. Superposition of the sequences of the fragments onto the three-dimensional allergen structure indicated that the removal of only one of the four helices had led to the destabilization of the alpha helical structure of Phl p 6. The lack of structural fold was accompanied by a strong reduction of IgE reactivity and allergenic activity of the three fragments as determined by basophil histamine release in allergic patients. Each of the three Phl p 6 fragments adsorbed to CFA induced Phl p 6-specific IgG Abs in rabbits. However, immunization of mice with fragments adsorbed to an adjuvant allowed for human use (AluGel-S) showed that only the Phl p 6 aa 31-110 induced Phl p 6-specific IgG Abs. Anti-Phl p 6 IgG Abs induced by vaccination with Phl p 6 aa 31-110 inhibited patients' IgE reactivity to the wild-type allergen as well as Phl p 6-induced basophil degranulation. Our results are of importance for the design of hypoallergenic allergy vaccines. They show that it has to be demonstrated that the hypoallergenic derivative induces a robust IgG response in a formulation that can be used in allergic patients.
Assuntos
Alérgenos/biossíntese , Alérgenos/genética , Regulação para Baixo/imunologia , Proteínas de Plantas/síntese química , Proteínas de Plantas/genética , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/genética , Vacinas/genética , Alérgenos/administração & dosagem , Alérgenos/imunologia , Animais , Regulação para Baixo/genética , Feminino , Regulação da Expressão Gênica/imunologia , Humanos , Soros Imunes/biossíntese , Imunoglobulina E/metabolismo , Imunoglobulina G/biossíntese , Imunoglobulina G/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Phleum/genética , Phleum/imunologia , Proteínas de Plantas/administração & dosagem , Proteínas de Plantas/imunologia , Pólen/genética , Pólen/imunologia , Engenharia de Proteínas/métodos , Dobramento de Proteína , Estrutura Secundária de Proteína , Coelhos , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/imunologia , Vacinas/administração & dosagem , Vacinas/síntese química , Vacinas/imunologiaRESUMO
BACKGROUND: Grasses belong to the most potent allergen sources worldwide. Group 2 grass pollen allergens are recognized by more than 100 million allergic patients. OBJECTIVE: The aim was to develop an assay for the specific detection and quantification of group 2 grass pollen allergens. METHODS: We have isolated a monoclonal human IgE Fab specific for group 2 grass pollen allergens by combinatorial cloning from lymphocytes of a grass pollen-allergic patient. This Fab was converted into a complete human IgG1 antibody and used together with rPh1 p 2 to develop a competitive ELISA for the specific measurement of group 2 allergens. ELISA plate-bound purified recombinant human Ph1 p 2-specific IgG1 is incubated with a constant amount of biotinylated rPh1 p 2 competing with increasing concentrations of group 2 allergens to be determined. Defined concentrations of purified rPhl p 2 are used to establish a standard curve. The concentration of unlabeled group 2 allergens can thus be deduced from the displacement of biotinylated rPh1 p 2, which can be detected with peroxidase-labeled streptavidin. RESULTS: The competition-ELISA measured rPh1 p 2 concentrations ranging from 10 ng/mL to 500 ng/mL and allowed to quantify group 2 allergens from 9 different grass families. The results were in good agreement with immunoblot data. CONCLUSIONS: The described assay can be used for standardization of diagnostic and therapeutic vaccines as well as for the quantification of group 2 allergens in environmental samples.
Assuntos
Alérgenos/análise , Anticorpos Monoclonais , Poaceae/imunologia , Pólen/imunologia , Alérgenos/classificação , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/estatística & dados numéricos , Humanos , Sensibilidade e Especificidade , Especificidade da EspécieRESUMO
Patch testing was done on 2776 consecutive patients (76.5% female) with a locally revised standard series of 34 contact allergens and the results analyzed for age- and gender-specific differences. At least one positive epicutaneous test reaction occurred in 48.9% of patients. Nickel (20.9%), ethylmercuric chloride (13.2%), thimerosal (11.8%), fragrance mix (9.3%), metallic mercury (8.9%), palladium (5.8%), balsam of Peru (3.8%), copper (3.7%), cobalt (3.3%), and chromium (2.3%) were the 10 most important sensitizers. The following tested allergens with sensitization rates of more than 1% were not part of the usual standard series: ethylmercuric chloride, metallic mercury, copper, propolis (1.3%), propylene glycol (1.0%). Reactions to nickel, cobalt, and palladium, but not to chromium, were significantly more abundant in females (p < 0.002, chi-squared test). The overall sensitization rate was highest in children less than 10 years old (62%) and decreased steadily, to be lowest among patients more than 70 years old (34.9%). The rate of positive reactions to nickel and thimerosal decreased with age, while fragrance mix and metallic mercury stayed at the same level through all age groups.
Assuntos
Alérgenos , Dermatite Alérgica de Contato/diagnóstico , Dermatite Alérgica de Contato/epidemiologia , Imunização/métodos , Adolescente , Adulto , Distribuição por Idade , Idoso , Idoso de 80 Anos ou mais , Áustria/epidemiologia , Criança , Pré-Escolar , Dermatite Alérgica de Contato/imunologia , Feminino , Humanos , Incidência , Masculino , Metais/farmacologia , Pessoa de Meia-Idade , Níquel/farmacologia , Testes do Emplastro/métodos , Perfumes/farmacologia , Sistema de Registros , Estudos Retrospectivos , Fatores de Risco , Distribuição por SexoRESUMO
BACKGROUND: Major allergens of oilseed rape (OSR) pollen with molecular weights of 6/8, 14 and between 27 and 69 kD have been described. The aim of the present study was to further characterize the 14-kD allergen. METHODS: The 14-kD protein was purified from OSR pollen extracts by poly-(L-proline) (PLP)-Sepharose affinity chromatography and characterized immunologically by means of allergic patients' IgE antibodies, profilin-specific rabbit antisera, Western blot and ELISA inhibition using recombinant birch profilin (rBet v 2), and skin prick testing. RESULTS: By PLP affinity chromatography, OSR pollen profilin was purified as a single protein of 14.5 kD and further identified as a profilin by three polyclonal rabbit antisera raised against ragweed and tobacco pollen profilin and the C-terminus of birch profilin. IgE binding of a human serum pool (n = 15) and four profilin-reactive sera to nitrocellulose-blotted OSR profilin was completely inhibited by 1 microg/ml rBet v 2 (birch profilin). Reciprocal ELISA inhibition using increasing concentrations of rBet v 2 and purified OSR profilin, respectively, showed that rBet v 2 strongly inhibits antibody binding to OSR profilin, whereas almost 100 times the amount of OSR profilin was needed to inhibit IgE binding to rBet v 2. Skin prick tests were positive (wheal >/=3 mm) with 5 microg/ml rBet v 2 in all three patients tested, and with OSR profilin in two patients at a concentration of 50 microg/ml. CONCLUSIONS: OSR pollen profilin shares IgE and IgG epitopes with Bet v 2 and other plant profilins and may represent a potentially relevant allergen for profilin-sensitized patients.
Assuntos
Brassica napus/imunologia , Proteínas Contráteis , Proteínas dos Microfilamentos/imunologia , Pólen/imunologia , Western Blotting , Cromatografia em Agarose , Reações Cruzadas/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina E/imunologia , Proteínas dos Microfilamentos/isolamento & purificação , Pólen/química , Profilinas , Testes CutâneosRESUMO
Histamine in food at non-toxic doses has been proposed to be a major cause of food intolerance causing symptoms like diarrhea, hypotension, headache, pruritus and flush ("histamine intolerance"). Histamine-rich foods such as cheese, sausages, sauerkraut, tuna, tomatoes, and alcoholic beverages may contain histamine up to 500 mg/kg. We conducted a randomized, double-blind, placebo-controlled cross-over study in 10 healthy females (age range 22-36 years, mean 29.1 +/- 5.4) who were hospitalized and challenged on two consecutive days with placebo (peppermint tea) or 75 mg of pure histamine (equaling 124 mg histamine dihydrochloride, dissolved in peppermint tea). Objective parameters (heart rate, blood pressure, skin temperature, peak flow) as well as a total clinical symptom score using a standardized protocol were recorded at baseline, 10, 20, 40, 80 minutes, and 24 hours. The subjects received a histamine-free diet also low in allergen 24 hours before hospitalization and over the whole observation period. Blood samples were drawn at baseline, 10, 20, 40, and 80 minutes, and histamine and the histamine-degrading enzyme diamine oxidase (DAO) were determined. After histamine challenge, 5 of 10 subjects showed no reaction. One individual experienced tachycardia, mild hypotension after 20 minutes, sneezing, itching of the nose, and rhinorrhea after 60 minutes. Four subjects experienced delayed symptoms like diarrhea (4x), flatulence (3x), headache (3x), pruritus (2x) and ocular symptoms (1x) starting 3 to 24 hours after provocation. No subject reacted to placebo. No changes were observed in histamine and DAO levels within the first 80 minutes in non-reactors as well as reactors. There was no difference in challenge with histamine versus challenge with placebo. We conclude that 75 mg of pure liquid oral histamine--a dose found in normal meals--can provoke immediate as well as delayed symptoms in 50% of healthy females without a history of food intolerance.
Assuntos
Histamina/efeitos adversos , Hipersensibilidade Tardia/etiologia , Hipersensibilidade Imediata/etiologia , Administração Oral , Adulto , Amina Oxidase (contendo Cobre)/metabolismo , Estudos Cross-Over , Método Duplo-Cego , Feminino , Histamina/administração & dosagem , Histamina/metabolismo , Humanos , Hipersensibilidade Tardia/sangue , Hipersensibilidade Tardia/enzimologia , Hipersensibilidade Imediata/sangue , Hipersensibilidade Imediata/enzimologia , Valores de ReferênciaRESUMO
The grass pollen allergen, Phl p 7, belongs to a family of highly cross-reactive calcium-binding pollen allergens. Because Phl p 7 contains most of the disease-eliciting epitopes of pollen-derived calcium-binding allergens, hypoallergenic variants were engineered according to the x-ray crystal structure of Phl p 7 for allergy vaccination. In three recombinant variants, amino acids essential for calcium binding were mutated, and two peptides comprising the N- and C-terminal half were obtained by synthetic peptide chemistry. As determined by circular dichroism analysis and size exclusion chromatography coupled to mass spectrometry, recombinant mutants showed altered structural fold and lacked calcium-binding capacity, whereas the two synthetic peptides had completely lost their structural fold. Allergic patients' IgE Ab binding was strongest reduced to the variant containing two mutations in each of the two calcium-binding sites and to the peptides. Basophil histamine release and skin test experiments in allergic patients identified the peptides as the vaccine candidates with lowest allergenic activity. Immunization of rabbits with the peptides induced IgG Abs that blocked allergic patients' IgE binding to Phl p 7 and inhibited allergen-induced basophil degranulation. Our results indicate that disruption of an allergen's three-dimensional structure represents a general strategy for the generation of hypoallergenic allergy vaccines, and demonstrate the importance of allergen-specific IgG Abs for the inhibition of immediate allergic symptoms.
Assuntos
Alérgenos/genética , Proteínas de Ligação ao Cálcio/genética , Dessensibilização Imunológica/métodos , Phleum/imunologia , Proteínas de Plantas/genética , Vacinas/síntese química , Vacinas/genética , Alérgenos/química , Alérgenos/imunologia , Alérgenos/metabolismo , Animais , Antialérgicos/administração & dosagem , Antialérgicos/síntese química , Antialérgicos/imunologia , Antígenos de Plantas , Basófilos/imunologia , Basófilos/metabolismo , Sítios de Ligação de Anticorpos/genética , Ligação Competitiva/imunologia , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/imunologia , Proteínas de Ligação ao Cálcio/metabolismo , Degranulação Celular , Linhagem Celular Tumoral , Reações Cruzadas , Relação Dose-Resposta Imunológica , Liberação de Histamina/imunologia , Humanos , Imunoglobulina E/metabolismo , Imunoglobulina G/biossíntese , Imunoglobulina G/metabolismo , Camundongos , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Phleum/química , Phleum/genética , Proteínas de Plantas/química , Proteínas de Plantas/imunologia , Proteínas de Plantas/metabolismo , Pólen/genética , Pólen/imunologia , Coelhos , Ratos , Testes Cutâneos , Relação Estrutura-Atividade , Vacinas/administração & dosagem , Vacinas/imunologiaRESUMO
BACKGROUND: Calcium-binding plant allergens can be grouped in different families according to the number of calcium-binding domains (EF hands). OBJECTIVE: We sought to identify pollens containing crossreactive calcium-binding allergens and to investigate structural and immunologic similarities of members belonging to different families of calcium-binding allergens. METHODS: By means of multiple sequence alignment and molecular modeling, we searched for structural similarities among pollen allergens with 2 (Phl p 7, timothy grass; Aln g 4, alder), 3 (Bet v 3, birch) and 4 EF hands (Jun o 4, prickly juniper). Purified recombinant Aln g 4 and Jun o 4 were used to determine the prevalence of IgE recognition in 210 patients sensitized to different pollens and to search, by means of ELISA competition, for the presence of cross-reactive epitopes in pollens from 16 unrelated plant species. IgE cross-reactivity among the allergen families was studied with purified rPhl p 7, rAln g 4, rBet v 3, and rJun o 4 and 2 synthetic peptides comprising the N-terminal and C-terminal EF hands of Phl p 7 by means of ELISA competition. RESULTS: Structural similarities were found by using molecular modeling among the allergens with 2, 3, and 4 EF hands. Pollens from 16 unrelated plants contained Aln g 4- and Jun o 4-related epitopes. Twenty-two percent of the patients with multiple pollen sensitization reacted to at least one of the calcium-binding allergens. A hierarchy of IgE cross-reactivity (rPhl p7 > rAln g 4 > rJun o 4 > rBet v 3) could be established that identified rPhl p 7 as the EF-hand allergen containing most IgE epitopes in the population studied. CONCLUSION: The demonstration that members of different families of calcium-binding plant allergens share similarities suggests that it may be possible to use representative molecules for the diagnosis and therapy of allergies to EF-hand allergens.