RESUMEN
BACKGROUND: Induction of allergen-specific IgG(4) antibodies is the most consistent immunological finding in immunotherapy trials. However, quantitative assessments of IgG(4) antibodies have not proven beneficial in evaluating clinical changes during or after immunotherapy. In the current study, we investigated the relationship between clinical outcome and allergen-specific IgG(4) titres or functional antibody responses following immunotherapy. We hypothesized that functional assays of serum IgG-associated inhibitory activity such as inhibition of IgE-allergen interactions (IgE-blocking factor) and inhibition of CD23-dependent IgE-facilitated allergen binding (IgE-FAB) correlate more closely with clinical outcome and may be biomarkers of clinical response. METHODS: In an 8-month dose-response randomized double-blind placebo-controlled study, 221 polysensitized subjects with severe seasonal rhinitis received Alutard SQ, Phleum pratense 100,000 SQ-U, 10,000 SQ-U or placebo injections. Serum specimens were collected before treatment, after up-dosing, during the peak season and at the end of the study. Allergen-specific IgG(4) titres and IgG-associated inhibitory activity were evaluated. RESULTS: A time- and dose-dependent increase in serum inhibitory activity for both the IgE-blocking factor and IgE-FAB was observed, which paralleled increases in grass pollen-specific IgG(4) antibodies. A modest but significant inverse relationship was demonstrated between postimmunotherapy serum inhibitory activity and combined symptom-rescue medication scores (IgE-FAB: r = -0.25, P = 0.0002; IgE-blocking factor: r = -0.28, P < 0.0001), whereas this was not observed for immunoreactive IgG(4) levels (r = -0.11, P = 0.12). CONCLUSIONS: Functional assays of inhibitory IgG(4) and IgE-blocking factor may be more useful surrogates of clinical response than IgG(4). Whether these antibody effects may serve as predictive biomarkers of clinical efficacy in individual patients requires further investigation.
Asunto(s)
Alérgenos/inmunología , Desensibilización Inmunológica , Inmunoglobulina G/inmunología , Phleum/inmunología , Polen/inmunología , Rinitis Alérgica Estacional/inmunología , Rinitis Alérgica Estacional/terapia , Alérgenos/administración & dosificación , Relación Dosis-Respuesta Inmunológica , Glicoproteínas/sangre , Glicoproteínas/inmunología , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Inmunoglobulina G/sangre , Inyecciones Subcutáneas , Proteínas de Neoplasias , Resultado del TratamientoRESUMEN
BACKGROUND: Specific immunotherapy is the only causal treatment of allergy available today. Traditionally, therapeutic products based on either a single grass species or a mix of such extracts are used for grass pollen immunotherapy. Investigations comparing the immunological response to these allergen preparations are needed to ensure optimal treatment. The objective of this study was to investigate patterns of T and B cell cross-reactivity to Pooideae single-species extracts and to extract mixes. METHODS: IgG4 induced by immunotherapy with Phleum pratense extract was investigated for cross-reactivity using nine single-species extracts and four mixes. For the mixes, studies of IgE cross-reactivity were also performed. T cell cross-reactivity was investigated in lines specific to nPhl p 1 or nPhl p 5 allergens, and the amounts of group 1 and 5 allergens in the extracts were quantified by a single radial immunodiffusion. RESULTS: The levels of treatment-induced IgG4 detected by all the extracts displayed a clear correlation to that detected by the P. pratense pollen extract. The IgE studies confirmed the cross-reactivity of P. pratense-specific B cells towards the allergens contained in the mixes, and the T cell studies demonstrated cross-reactivity towards group 1 and 5 major allergens in extracts of six temperate grass species. CONCLUSION: Extensive T and B cell cross-reactivity was observed towards the allergens of the Pooideae grasses, and the degree of B cell cross-reactivity was independent of the number of species included in the extract mixes. This implies that treatment with pollen extract of just one Pooideae species will affect the allergic responses caused by any of the temperate grasses in this subfamily.
Asunto(s)
Linfocitos B/inmunología , Desensibilización Inmunológica , Hipersensibilidad/terapia , Phleum/inmunología , Polen/inmunología , Linfocitos T/inmunología , Línea Celular , Niño , Preescolar , Reacciones Cruzadas/inmunología , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , Extractos Vegetales/inmunología , Extractos Vegetales/uso terapéutico , Poaceae/inmunologíaRESUMEN
Immunotherapy is the only treatment for allergy that has the potential to alter the natural course of the disease. Sublingual immunotherapy for grass pollen-induced rhinoconjunctivitis has been developed to make immunotherapy available to a broader group of allergic patients. Here, a safe dose range and the safety during daily sublingual administration were investigated for a new tablet-based sublingual immunotherapy for grass pollen allergy. Simultaneously, immunological changes were monitored. A randomized, double-blind, placebo-controlled phase I trial was undertaken, with stepwise dose-escalation during the dose-finding period, and afterwards with daily dosing 8 weeks prior to and 15 weeks during the grass pollen season (2500, 25000, or 75000 standardized quality tablet [SQ-T] units, or placebo). Fifty-two participants with grass pollen-induced rhinoconjunctivitis and a positive skin prick test and specific IgE to Phleum pratense entered the trial. During the daily-dose treatment periods, 67% of the participants reported adverse events. The most frequent were itching in the mouth, eyes, or throat, and rhinitis, and most were mild and resolved within 1 day. Two participants withdrew due to adverse events (sting and blisters in the mouth and itching in the mouth). Time- and dose-dependent increases of P pratense-specific IgG, IgA, IgE, and IgE-competing components were found in serum during the first 8 weeks of daily dosing, indicating that the treatment had a significant allergen-specific effect on the immune system. In conclusion, the grass allergen tablet, administered in a dose of 75000 SQ-T once daily, was well tolerated and displayed systemic immunogenicity.
Asunto(s)
Alérgenos/administración & dosificación , Inmunoterapia/métodos , Poaceae/inmunología , Polen/inmunología , Rinitis Alérgica Estacional/terapia , Administración Oral , Administración Sublingual , Adolescente , Adulto , Anciano , Alérgenos/inmunología , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Humanos , Hipersensibilidad/inmunología , Hipersensibilidad/terapia , Inmunoglobulinas/inmunología , Persona de Mediana Edad , Rinitis Alérgica Estacional/inmunología , Comprimidos , Factores de Tiempo , Resultado del TratamientoRESUMEN
BACKGROUND: The clinical efficacy of specific allergy vaccination (SAV), previously called specific immunotherapy is well documented. The working mechanism of this treatment is not completely known at present. Allergen-specific CD4+ T lymphocytes are activated at extremely low allergen concentrations in vivo possibly as a result of serum IgE-facilitated allergen presentation (S-FAP). Previously, we have shown that this process can be inhibited by long-term birch SAV sera. METHODS: In the present study, we have analysed sera from birch-allergic patients in a randomized double-blind, placebo-controlled clinical trial for their ability to mediate S-FAP. Birch-specific IgE levels were not changed after SAV. Bet v 1-specific IgE levels, however, were significantly decreased (P<0.05) and Bet v 1-specific IgG4 levels were strongly increased after SAV (P<0.001). None of these changes were observed in the placebo group. When the sera were tested for their ability to induce S-FAP, a complete abrogation of this effect was noted in the sera from patients receiving active treatment (P<0.001), but not in the control group. This inhibition of S-FAP seemed to be associated with the reduction in the ratio between Bet v 1-specific IgE and IgG4 antibodies in serum, but a clear correlation could not be demonstrated. CONCLUSION: In conclusion, the present study clearly shows that SAV leads to an inhibition of the S-FAP needed to obtain optimal T cell activation at the low allergen concentrations present in vivo. This novel mechanism may explain the increased allergen threshold levels found in allergen provocation tests and the reduction of late-phase reactions observed after SAV.
Asunto(s)
Presentación de Antígeno , Betula , Desensibilización Inmunológica/métodos , Inmunoglobulina E/inmunología , Receptores de IgE/inmunología , Rinitis Alérgica Estacional/inmunología , Alérgenos/inmunología , Antígenos de Plantas , Células Cultivadas , Método Doble Ciego , Humanos , Inmunoglobulina G/inmunología , Activación de Linfocitos , Polen , Estadísticas no Paramétricas , Linfocitos T/inmunologíaRESUMEN
The symptoms characteristic of allergic hypersensitivity are caused by the release of mediators, i.e., histamine, from effector cells such as basophils and mast cells. Allergens with more than one B cell epitope cross-link IgE Abs bound to high affinity FcepsilonRI receptors on mast cell surfaces leading to aggregation and subsequent mediator release. Thus, allergen-Ab complexes play a crucial role in the cascade leading to the allergic response. We here report the structure of a 1:1 complex between the major birch pollen allergen Bet v 1 and the Fab fragment from a murine monoclonal IgG1 Ab, BV16, that has been solved to 2.9 A resolution by x-ray diffraction. The mAb is shown to inhibit the binding of allergic patients' IgE to Bet v 1, and the allergen-IgG complex may therefore serve as a model for the study of allergen-IgE interactions relevant in allergy. The size of the BV16 epitope is 931 A2 as defined by the Bet v 1 Ab interaction surface. Molecular interactions predicted to occur in the interface are likewise in agreement with earlier observations on Ag-Ab complexes. The epitope is formed by amino acids that are conserved among major allergens from related species within the Fagales order. In combination with a surprisingly high inhibitory capacity of BV16 with respect to allergic patients' serum IgE binding to Bet v 1, these observations provide experimental support for the proposal of dominant IgE epitopes located in the conserved surface areas. This model will facilitate the development of new and safer vaccines for allergen immunotherapy in the form of mutated allergens.
Asunto(s)
Alérgenos/metabolismo , Anticuerpos Monoclonales/metabolismo , Epítopos Inmunodominantes/metabolismo , Fragmentos Fab de Inmunoglobulinas/metabolismo , Inmunoglobulina G/metabolismo , Proteínas de Plantas/metabolismo , Polen/inmunología , Rinitis Alérgica Estacional/metabolismo , Alérgenos/química , Alérgenos/inmunología , Animales , Anticuerpos Monoclonales/química , Especificidad de Anticuerpos , Antígenos de Plantas , Simulación por Computador , Reacciones Cruzadas , Humanos , Epítopos Inmunodominantes/química , Epítopos Inmunodominantes/inmunología , Fragmentos Fab de Inmunoglobulinas/química , Ratones , Modelos Moleculares , Proteínas de Plantas/química , Proteínas de Plantas/inmunología , Rinitis Alérgica Estacional/inmunología , Rosales , ÁrbolesRESUMEN
BACKGROUND: The role of allergen-specific CD4+ T lymphocytes in the pathophysiology of atopic disease is well established. Previous studies on allergen-specific T-cell responses have focused on the recognition of single major allergens to identify T-cell epitopes. OBJECTIVE: However, it is not clear whether immune responses to allergen extracts are exclusively targeted at major allergens or whether additional proteins are recognized. METHODS: Here we describe the Phleum pratense-specific immunoglobulin E (IgE) and T-cell responses of six allergic rhinitis patients. Reactivity was measured to size-separated fractions of a P. pratense extract as well as to the purified major allergens Phl p 1, Phl p 2/3 and Phl p 5. RESULTS: The specificity of the patients' serum IgE, measured in a fluid phase assay, was restricted to one or two of the major allergens. Even though the majority of the patients had IgE antibodies reactive with a single major allergen, one patient reacted with both Phl p 5 and with Phl p 2/3. Analysis of the T-cell repertoire with P. pratense-specific T-cell lines (TCLs) and CD4+ T-cell clones (TCCs) revealed that at least six different proteins were recognized, including the three major allergens, most notably Phl p 5. Simultaneous production of IL-5 and interferon (IFN) -gamma was detected in supernatants of the TCLs stimulated with P. pratense extract and the major allergens. CONCLUSION: These results indicate that allergic rhinitis patients have a large pool of circulating allergen-specific CD4+ T cells that recognize many different proteins in an allergenic extract, whereas only a small number of these proteins are recognized by serum IgE.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Inmunoglobulina E/inmunología , Proteínas de Plantas/inmunología , Poaceae/inmunología , Rinitis Alérgica Estacional/inmunología , Alérgenos/inmunología , Línea Celular , Cromatografía en Gel/métodos , Citocinas/metabolismo , Humanos , Inmunoglobulina E/sangre , Polen/inmunologíaRESUMEN
BACKGROUND: The involvement of CD4+ T cells in the pathophysiology of atopic disease is well established. OBJECTIVE: To gain further insight into the activation requirements for allergen-specific T cells, we characterized epitope specificity, HLA restriction and T-cell receptor (TCR) usage for T cells specific to Phl p 5, the group 5 major allergen of the grass Phleum pratense. METHODS: To identify the T-cell epitopes of Phl p 5, three Phl p 5-specific T-cell lines (TCLs) and 15 T-cell clones (TCCs) generated from the peripheral blood of three grass-allergic patients were tested with recombinant truncated Phl p 5a fragments and synthetic Phl p 5b peptides representing these two different recombinant Phl p 5 isoallergens. Additional activation experiments with HLA-subtyped antigen-presenting cells and flow cytometry analysis with TCR V-specific mAb were performed to further characterize the activation requirements for Phl p 5-specific TCCs. RESULTS: At least nine distinct T-cell specificities were identified and the T-cell epitopes recognized differed considerably among the three patients. Most of the epitopes found were isoform-specific, whereas three epitopes were shared between Phl p 5a and 5b. Several human leucocyte antigen (HLA) class II molecules were involved in the recognition of Phl p 5. Different HLA restriction specificities were even found among TCCs specific to the same epitope region. All TCCs were TCR-alpha/beta positive, and an overrepresentation of TCR Vbeta 3.1+ clones among TCCs specific to Phl p 5 appear to exists as 31% (4/13) of the TCCs expressed TCR Vbeta 3.1 (compared with 5% TCR Vbeta 3.1+ T cells in human peripheral blood) with no correlation with epitope specificity or HLA restriction. CONCLUSION: The T-cell reactivity of the three grass-allergic patients investigated shows that isoallergen-specific T-cell epitopes are found throughout the peptide backbone of Phl p 5a and Phl p 5b, and dominant T-cell epitopes of Phl p 5 were not identified. This indicates that a mixture of at least full-length rPhl p 5a and rPhl p 5b may be required to target the total Phl p 5-specific T-cell response of atopic patients.
Asunto(s)
Alérgenos/inmunología , Epítopos de Linfocito T/inmunología , Proteínas de Plantas/inmunología , Poaceae/inmunología , Polen/inmunología , Alérgenos/química , Alérgenos/genética , Secuencia de Aminoácidos , Donantes de Sangre , Células Cultivadas , Células Clonales , Antígenos HLA/inmunología , Antígenos HLA/metabolismo , Humanos , Datos de Secuencia Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Isoformas de Proteínas , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Linfocitos T/inmunologíaAsunto(s)
Alérgenos/química , Epítopos/química , Polen/química , Árboles/química , Animales , Anticuerpos Monoclonales/inmunología , Antígenos de Plantas , Reacciones Cruzadas/inmunología , Epítopos/inmunología , Ratones , Proteínas de Plantas/química , Proteínas de Plantas/genética , Mutación Puntual/inmunología , Polen/inmunología , Conformación Proteica , Difracción de Rayos XRESUMEN
BACKGROUND: At present, recombinant allergens are considered for the diagnosis and treatment of atopic allergies. To evaluate the theoretical impact of isoallergen variation on the selection of isoallergens for specific allergy vaccination, we characterized the T-cell response of allergic patients to the Phleum pratense major allergen, Phl p 5, and five of its recombinant isoallergens. METHODS: Phl p 5-specific T cell lines (TCLs) were isolated from the peripheral blood of 3 allergic rhinitis patients, and their reactivity patterns were studied in detail. RESULTS: The TCLs were highly crossreactive with related grasses. The crossreactivity with Poa pratensis was more extensive than with Lolium perenne, directly reflecting the sequence identity between Phl p 5, Poa p 5, and Lol p 5. The TCLs produced IFN-gamma and IL-4 simultaneously, resembling a Th0-like cytokine production profile. Interestingly, when T cell clones were tested with natural Phl p 5 and five rPhl p 5 isoallergens, a differential recognition pattern was found. One of the TCLs exclusively reacted with Phl p 5b, another reacted with all isoforms tested, and the third reacted strongly with native purified Phl p 5, but only weakly with all five recombinant isoallergens. CONCLUSION: These results indicate that Phl p 5-specific T cells are highly heterogeneous, and that they differentially recognize isoallergens. This indicates that when recombinant Phl p 5 is considered for allergy vaccination, a mixture of isoallergens representing the different isoallergen groups may clinically prove to be more effective than single isoallergens.
Asunto(s)
Alérgenos , Hipersensibilidad/inmunología , Proteínas de Plantas/inmunología , Ribonucleasas/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Presentación de Antígeno , Humanos , Epítopos Inmunodominantes , Isoantígenos/inmunología , Datos de Secuencia Molecular , Proteínas de Plantas/genética , Polen , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Ribonucleasas/genética , Alineación de SecuenciaRESUMEN
The human type I allergic response is characterized by the presence of allergen-specific serum immunoglobulin E (IgE). Allergen-mediated cross-linking of receptor-bound IgE on the surface of mast cells and circulating basophils triggers the release of mediators, resulting in the development of the clinical symptoms of allergy. In order to study the structural basis of allergen-antibody interaction, a complex between the major birch-pollen allergen Bet v 1 and a Fab' fragment isolated from the murine monoclonal Bet v 1 antibody BV16 has been crystallized. Complex crystals belong to space group P1, with unit-cell parameters a = 91.65, b = 99.14, c = 108.90 A, alpha = 105.7, beta = 98.32, gamma = 97.62 degrees, and diffract to 2.9 A resolution when analyzed at 100 K using synchrotron-generated X--rays.
Asunto(s)
Alérgenos/química , Alérgenos/aislamiento & purificación , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Polen/química , Animales , Anticuerpos Monoclonales/química , Reacciones Antígeno-Anticuerpo , Antígenos de Plantas , Cristalización , Cristalografía por Rayos X , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Inmunoglobulina G/química , Ratones , Polen/inmunología , Rinitis Alérgica Estacional/inmunologíaRESUMEN
BACKGROUND: Pollinosis, caused by grasses of the Poaceae family, is a problem worldwide. The relative importance of grass groups 1 and 5 major allergens is well established. However, not much is known about the recognition of these allergens by T cells and whether this T-cell reactivity correlates with skin reactivity and serum IgE levels. OBJECTIVES: The aim of this study was to characterize the cross-reactive, grass allergen-specific T-cell responses from patients allergic to grass and nonatopic individuals and to investigate whether these responses correlate with grass-specific IgE and skin reactivity. METHODS: Skin prick test wheal areas and grass-specific serum IgE levels were determined in all patients (n = 21) and nonallergic control donors (n = 20). Peripheral blood mononuclear cells were isolated and stimulated with grass allergen extracts (Phleum pratense, Poa pratensis, Lolium perenne) and immunoaffinity-purified group 5 allergens, and the production of type 1 and type 2 cytokines was determined in the patient group. RESULTS: Donors allergic to grass showed increased T-cell-proliferative responses to grass allergens compared with nonatopic control subjects. We find it interesting that the magnitude of the patients' T-cell responses could not be correlated with the individual skin prick test areas and specific serum IgE levels, and several patients with allergies to grass had group 5-specific T-cell responses in the absence of group 5-specific IgE. The absence of a correlation between T-cell proliferation and IgE levels or skin prick test results may in part be explained by the finding that patients predominantly produced IL-5 in response to Phl p 5, the major allergen, and predominantly IFN-gamma in response to Phl p extract. In general, all donors responded equally well to all three grasses. Additional experiments with Phl p 5-specific T-cell lines indicated that the equal proliferation of peripheral blood mononuclear cells to all three species is the direct result of cross-reactivity. CONCLUSIONS: Grass allergen-specific T-cell responses are highly cross-reactive, and patients with allergies exhibit higher responses than nonallergic donors, suggesting that T cells are involved in the allergic reaction to grass group 5 allergens. However, group 5-specific T-cell responses are also found in donors without group 5-specific IgE, and the patients' grass-specific T-cell responses and cytokine production do not correlate to skin reactivity or to concentrations of grass-specific IgE.
Asunto(s)
Alérgenos/inmunología , Hipersensibilidad/inmunología , Inmunoglobulina E/sangre , Activación de Linfocitos/inmunología , Poaceae/inmunología , Polen/inmunología , Linfocitos T/inmunología , Alérgenos/efectos adversos , Reacciones Cruzadas , Citocinas/biosíntesis , Humanos , Hipersensibilidad/sangre , Inmunoglobulina E/inmunología , Fenotipo , Proteínas de Plantas/inmunología , Pruebas CutáneasRESUMEN
The long-term effect of tree pollen extract immunotherapy was investigated 6 years after termination of the treatment. Subjective symptom evaluation of 36 patients 6 years after a 3-year period of immunotherapy showed that rhinitis and asthma symptoms remained at the improved level reached just after termination of the treatment. Some 86% of the rhinitis patients and 68% of the asthma patients maintained improvement. None of the rhinitis patients developed asthma in the study period. Skin prick tests reflected the outcome of the subjective symptom assessment. The skin sensitivity of the patients decreased significantly during immunotherapy, and the skin reactions 6 years after specific immunotherapy were still significantly lower than the pretreatment levels. Total IgE and birch-specific IgE levels were constant throughout the study period, and both the affinity and epitope specificity of the IgE antibodies of the patients were the same before, during, and 6 years after treatment. In conclusion, specific immunotherapy reduces symptoms in patients suffering from rhinitis and asthma, and the effect is maintained 6 years after termination of the treatment. Specific immunotherapy seems to prevent long-term development of asthma in rhinitis patients. IgE measurements do not reflect the overall status of the patients.
Asunto(s)
Alérgenos , Asma/prevención & control , Desensibilización Inmunológica/normas , Polen , Rinitis Alérgica Estacional/prevención & control , Asma/inmunología , Desensibilización Inmunológica/métodos , Estudios de Seguimiento , Humanos , Inmunoglobulina E/sangre , Rinitis Alérgica Estacional/inmunología , Pruebas Cutáneas , Factores de Tiempo , Resultado del Tratamiento , ÁrbolesRESUMEN
A solid-phase, monoclonal antibody-based ELISA was set up to quantitate group 5 allergens in pollen extracts of wild and cultivated Pooideae grasses. The method was able to evaluate group 5 concentration in mass units with a sensitivity in the ng/ml range and a practical working range of 1-100 ng/ml. The group 5 ELISA was compared with rocket immunoelectrophoresis for determination of allergen levels in several Phleum pratense extracts, and a very good quantitative correlation was found (r = 0.98; P < 0.0001). A highly significant correlation (r > 0.8) was also obtained in comparing allergenic potency determined by RAST inhibition to group 5 content in several wild and cultivated grass species. The results proved the usefulness of the method in the standardization of Pooideae pollen extracts employed in diagnosis and treatment.
Asunto(s)
Alérgenos/inmunología , Antígenos/análisis , Ensayo de Inmunoadsorción Enzimática/métodos , Poaceae/inmunología , Alérgenos/aislamiento & purificación , Anticuerpos Monoclonales/inmunología , Relación Dosis-Respuesta Inmunológica , Inmunización/normas , Immunoblotting , Inmunoelectroforesis , Inmunoglobulina E/análisis , Polen/inmunología , Prueba de RadioalergoadsorciónRESUMEN
BACKGROUND: Fagales pollens are considered among the main agents responsible for allergic diseases in many countries of the northern hemisphere and single major allergens have been shown to be responsible for these responses. OBJECTIVE: To develop a solid phase immunoassay for the quantification of Bet v 1, the main allergen from Betula verrucosa (birch), and to assess its suitability for quantitating the equivalent major allergen in other Fagales species as well. METHODS: The assay is based on the use of two different anti-Bet v 1 monoclonal antibodies which were immobilized on the solid phase and, as a primary standard, affinity purified Bet v 1, the protein content of which was determined by amino acid analysis. RESULTS: The ELISA proved to measure less than 0.2 ng/mL of Bet v 1 with a practical range of 0.4-40 ng/mL and could be suitable to quantify the equivalent major allergen in other Fagales species such as Corylus avellana (hazel), Carpinus betulus (horbeam) and Alnus glutinosa (alder). The method was compared with quantitative electrophoresis and rocket immuno-electrophoresis for the determination of the allergen content in several Betula verrucosa extracts, and a very good quantitative correlation was found. Likewise, the Bet v 1 content exhibited a good correlation (r = 0.87; P < 0.005) with the allergenic potency values obtained by RAST inhibition. CONCLUSIONS: The results indicate that the Bet v 1-assay could be useful for standardization purposes in Fagales pollen extracts intended for clinical use.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Proteínas de Plantas/análisis , Polen/química , Alérgenos/análisis , Anticuerpos Monoclonales , Antígenos de Plantas , Electroforesis en Gel de Poliacrilamida/métodos , Ensayo de Inmunoadsorción Enzimática/normas , Humanos , Inmunoensayo , Inmunoelectroforesis/métodos , Inmunoglobulina E/inmunología , Inmunoglobulina E/metabolismo , Proteínas de Plantas/inmunología , Proteínas de Plantas/aislamiento & purificación , Polen/inmunología , Prueba de Radioalergoadsorción , Sensibilidad y Especificidad , ÁrbolesRESUMEN
BACKGROUND: Allergen-specific T lymphocytes play an important role in the pathophysiology of atopic disease. Detailed studies of their epitope-specificity and crossreactivity are required for the development of novel approaches for specific immunotherapy. OBJECTIVES: The aim of the study was to characterize the fine specificity of Bet v 1-specific T cells from allergic donors. METHODS: Polyclonal T-cell lines (TCL) and T-cell clones (TCC), specific for Bet v 1, the major birch (Betula verrucosa) pollen allergen, were isolated from the peripheral blood of three birch-allergic patients. Their epitope-specificity was studied using overlapping synthetic peptides, and crossreactivity with other tree pollen allergens of the Fagales order was evaluated. In addition, the Bet v 1-specific TCC were studied for their phenotype and cytokine production. RESULTS: All isolated Bet v 1-specific TCC (19/21 CD4+, 2/21 CD8+) reacted with affinity purified Bet v 1, but showed different reactivities with recombinant Bet v 1 (rBet v 1), and with group 1 allergens from other Fagales species. Epitope mapping of rBet v 1-reactive TCC with synthetic peptides of Bet v 1 showed the presence of four T-cell epitopes. Polyclonal T-cell lines reacted with 13 different peptides, and displayed even broader crossreactivity with group 1 pollen allergens from other Fagales members. CONCLUSION: This study demonstrates that apart from T-cell epitopes of rBet v 1, many other crossreactive or Bet v 1 isoallergen-specific epitopes exist. This indicates that isoallergenic variation plays an important role in the induction of Bet v 1-specific and crossreactive T-cell responsiveness to allergens.
Asunto(s)
Alérgenos/inmunología , Epítopos/inmunología , Inmunización , Proteínas de Plantas/inmunología , Polen/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Antígenos de Plantas , Línea Celular/inmunología , Células Clonales/inmunología , Reacciones Cruzadas/inmunología , Epítopos/química , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Datos de Secuencia Molecular , Oligopéptidos/síntesis química , Oligopéptidos/inmunología , Mapeo Peptídico , Proteínas de Plantas/análisis , Polen/química , Proteínas Recombinantes/inmunología , Sensibilidad y Especificidad , Homología de Secuencia de Aminoácido , ÁrbolesRESUMEN
Japanese cedar (Cryptomeria japonica) possibilities has been a serious allergic disease in Japan. There are two kinds of Japanese cedar trees; the popular one is diploid, the less popular is triploid. These trees are not very different morphologically. However, the relative allergenicity of their pollens is unknown, although both major allergens, Cry j 1 and Cry j 22, have been purified and cloned from the diploid line. Triploid trees are sterile and the allergenicity of their pollen may differ. Using Japanese-cedar-allergic patient sera, we compared the concentration of these two major allergens in both kinds of pollen. Pollen collected from different years and regions was also studied. The results indicate that triploid tree pollen extract has lower concentrations of both major allergens; therefore, the pollen may be less allergenic.
Asunto(s)
Alérgenos , Diploidia , Hipersensibilidad Inmediata/etiología , Extractos Vegetales/análisis , Proteínas de Plantas/análisis , Poliploidía , Árboles/clasificación , Antígenos de Plantas , Estudios de Casos y Controles , Humanos , Japón , Proteínas de Plantas/efectos adversos , Proteínas de Plantas/química , Características de la Residencia , Árboles/genéticaRESUMEN
The three-dimensional structure of the major birch pollen allergen, the 17,500 M(r) acidic protein Bet v 1 (from the birch, Betula verrucosa), is presented as determined both in the crystalline state by X-ray diffraction and in solution by nuclear magnetic resonance (NMR) spectroscopy. This is the first experimentally determined structure of a clinically important inhalant major allergen, estimated to cause allergy in 5-10 million individuals worldwide. The structure shows three regions on the molecular surface predicted to harbour cross-reactive B-cell epitopes which provide a structural basis for the allergic symptoms that birch pollen allergic patients show when they encounter pollens from related trees such as hazel, alder and hornbeam. The structure also shows an unusual feature, a 30 A-long forked cavity that penetrates the entire protein.
Asunto(s)
Alérgenos/química , Proteínas de Plantas/química , Polen/química , Secuencia de Aminoácidos , Antígenos de Plantas , Cristalografía por Rayos X , Epítopos de Linfocito T/química , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación ProteicaRESUMEN
A gene encoding the pollen major allergen Bet v 1 from Betula verrucosa (White Birch) has been cloned and expressed in Escherichia coli as a fusion with maltose-binding protein and a Factor Xa proteolytic cleavage site. A generally applicable cloning strategy based on polymerase chain reaction was designed to position the Factor Xa proteolytic site so that the authentic amino terminus of Bet v 1 was generated after cleavage. Fusion protein was isolated by amylose affinity chromatography and enzymatically cleaved by incubation with Factor Xa. Recombinant Bet v 1 was isolated by gel filtration and gave rise to a single band with apparent molecular weight of 17 kDa when analyzed by SDS-polyacrylamide gel electrophoresis. N-terminal sequencing of the first 20 amino acids showed complete agreement with the deduced Bet v 1 DNA sequence. Mass spectrometry showed that recombinant Bet v 1 has a molecular mass of 17,440 +/- 2 Da; 86% of the recombinant Bet v 1 amino acid sequence could be verified by digestion with Lys-C and mass spectrometric peptide mapping. The yield of purified recombinant Bet v 1 was 10 mg per liter E. coli cell culture. Two-dimensional gel electrophoresis of purified recombinant protein gave rise to one major protein spot and one or two minor spots focusing at slightly different pHs. The immunochemical properties of recombinant protein were indistinguishable from those of naturally occurring Bet v 1 when compared using a panel of murine monoclonal antibodies and serum IgE from birch pollen allergic patients. Furthermore, recombinant Bet v 1 elicited T-cell proliferation comparable to that of natural Bet v 1. Thus, the methods used for bacterial expression and protein purification result in relatively high yields of folded recombinant Bet v 1 with correct N-terminal sequence and molecular mass. Furthermore, the B- and T-cell epitope structures of recombinant Bet v 1 closely resemble those of the natural protein from pollen.