Fractionation and analysis of allergenicity of allergens from Prosopis juliflora pollen

Prosopis juliflora pollen allergen extract was prepared, and its crude allergen extract was fractionated by Sephadex G-100 gel filtration. Six different fractions were obtained which was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Protein and carbohydrate content of each fraction were estimated. Fraction E (MW 20,000) showed a 25% carbohydrate concentration. The amino acid analysis indicated that this fraction was rich in glutamic acid and alanine. Antigenicity or allergenicity of fractionated allergens were checked by gel diffusion test, rocket immunoelectrophoresis, skin prick test, and radioallergosorbent test. All these test indicate that fraction E consisted mainly of allergenic molecules (MW 20,000) of P. juliflora pollen.     

Studies on `allergoids' prepared from naturally occurring allergens: I. Assay of allergenicity and antigenicity of formalinized rye Group I component*

The highly purified major allergenic component of rye grass pollen (Group I) was used to investigate the possibility of destroying selectively the allergenic properties of an antigen, while largely retaining its original immunizing capacities. The allergen was treated under mild conditions with formalin alone or formalin plus a reactive low molecular weight additive. Certain derivatives (allergoids) showed well over 99 per cent reduction in allergenicity, determined by the histamine released from allergic human leucocytes in vitro, but were still able to combine with rabbit antibody against native antigen. Furthermore, the allergoids stimulated production (in guinea-pigs) of appreciable amounts of antibody able to inhibit native allergen-mediated human allergic histamine release in vitro and to cross-react with native antigen by PCA tests in normal guinea-pigs.

Residual allergenicity and cross-immunogenicity (by the inhibition assay) of the different formalinized derivatives varied appreciably according to the additive used in formalinization, but the cross-reactivities of the different preparations in quantitative precipitin analysis against rabbit anti-native antigen serum were similar. The residual allergenicities of individual derivatives varied by up to 1000-fold in different cell preparations, suggesting a heterogeneity of allergenic determinants. Allergoid derivatives showed no hapten-like activity in that they were unable to inhibit allergen-mediated histamine release from leucocytes.

The theoretical and practical application of allergoids is discussed, including their potential usefulness in improving the immunotheraphy of atopic humans.

     

Allergenicity, immunogenicity and dose-relationship of three intact allergen vaccines and four allergoid vaccines for subcutaneous grass pollen immunotherapy

Different vaccines containing intact allergens or chemically modified allergoids as active ingredients are commercially available for specific immunotherapy. Allergoids are claimed to have decreased allergenicity without loss of immunogenicity and this is stated to allow administration of high allergoid doses. We compared the allergenicity and immunogenicity of four commercially available chemically modified grass pollen allergoid products with three commercially available intact grass pollen allergen vaccines. The allergenicity was investigated with immunoglobulin (Ig)E-inhibition and basophil activation assays. Human T cell proliferation and specific IgG-titres following mouse immunizations were used to address immunogenicity. Furthermore, intact allergen vaccines with different contents of active ingredients were selected to study the influence of the allergen dose. In general, a lower allergenicity for allergen vaccines was clearly linked to a reduced immunogenicity. Compared with the vaccine with the highest amount of intact allergen, the allergoids caused reduced basophil activation as well as diminished immunogenicity demonstrated by reduced T cell activation and/or reduced induction of murine grass-specific IgG antibodies. Interestingly, intact allergen vaccines with lower content of active ingredient exhibited similarly reduced allergenicity, while immunogenicity was still higher or equal to that of allergoids. The low allergenicity observed for some allergoids was inherently linked to a significantly lower immunogenic response questioning the rationale behind the chemical modification into allergoids. In addition, the linkage between allergenicity, immunogenicity and dose found for intact allergen vaccines and the immunogen as well as allergenic immune responses observed for allergoids suggest that the modified allergen vaccines do not contain high doses of immunologically active ingredients.     

Studies on “allergoids” prepared from naturally occurring allergens II. Evaluation of allergenicity and assay of antigenicity of formalinized mixed grass pollen extracts

A crude dialyzed mixed grass pollen extract was modified under mild conditions with formaldehyde in the presence or absence of low molecular weight additives which participate in the chemical reaction. By scratch and intradermal skin testing of grass-sensitive subjects, certain of the derivatives (allergoids) were found to be about 200 to 2,000 times less allergenically reactive than the native pollen extract (allergen). The allergoids largely retained their capacity to induce guinea pigs to produce antibody which strongly cross-reacted with the allergen, as measured by passive cutaneous anaphylaxis (PCA) in normal guinea pigs. Cross-antigenicity between the allergen and allergoids was also observed by immunodiffusion analysis with the use of antiallergen and antiallergoid sera. One of the formalinized derivatives also was studied by bronchial inhalation challenge experiments in 5 asthmatic subjects.     

Purification and partial characterization ofa 67-kD cross-react ive allergen from Imperata cylindrica pollen extract

BACKGROUND: Grass pollens are known to induce type I allergic reactions in a large number of genetically predisposed individuals. Earlier studies have recognized Imperata cylindrica (Ic) pollen as an important source of aeroallergen which contained 7 IgE binding proteins in the MW range of 85-16 kD. OBJECTIVES: To isolate, purify and characterize a cross-reactive allergenic protein from Ic pollen extract for diagnosis and therapy of grass pollen allergy. METHODOLOGY: Ic pollen extract was fractionated using DEAE Sephadex A-50, Sephadex G-200 and Mono Q column. Allergenic activity of the fractions was checked by ELISA, skin tests, ELISA inhibition and immunoblot using sera of Ic-sensitive patients. A 67-kD protein was purified to homogeneity from Ic-VIII. The allergenic determinants of this protein were identified by SDS-PAGE and immunoblot after CNBr treatment. RESULTS: Among Ic fractions, Ic-VIII was highly potent by ELISA, skin tests and showed cross-reactivity with 4 other tropical grasses by immunoblot and ELISA inhibition. The subfraction Ic-VIIIe1 of Ic-VIII showed a band at 67 kD on SDS-PAGE. On CNBr treatment, it gave 7 peptides, 3 of which were found to be allergenic. CONCLUSION: A 67-kD protein (Ic-VIIIe1) was isolated, purified to homogeneity and partially characterized. It showed cross-reactivity with tropical grasses tested and contained at least three allergenic determinants.     

Immunochemical characterization of a high molecular weight basic allergen (HMBA) of rye grass (Lolium perenne) pollen

A high molecular weight basic allergen (HMBA) was isolated from the mixture of non-dialysable components of the aqueous extract of defatted rye grass pollen by a combination of gel filtration and isoelectrofocussing. HMBA, a glycoprotein of mol. wt 56,800 (17% carbohydrate) contained all naturally occurring amino acids. A hyperimmune rabbit anti-HMBA serum gave only a single precipitin band with the crude extract of the rye grass pollen in crossed immunoelectrophoresis. Thus. it was concluded that HMBA was a unique and highly purified antigen. The allergenicity of HMBA was revealed by its ability to elicit immediate skin reactions in grass allergic patients. Moreover, all patients' sera tested had IgE antibodies to HMBA detectable by direct RAST with HMBA allergosorbent discs, These observations indicated that HMBA was a major allergenic constituent of rye grass pollen. Treatment of HMBA by 6 M guanidine HC1 led to a significant reduction in its ability to combine with human IgE antibodies. The treatment also resulted in the complete loss of allergenicity (i.e. inability to elicit PCA reactions with a murine reaginic antiserum to HMBA) and antigenicity (inability to form precipitins with rabbit anti-HMBA): hence, it would appear that the allergenic and antigenic determinants of HMBA are ‘conformational’.     

梭子蟹变应原的分离、纯化与鉴定

目的：对梭子蟹（Portunus pelogicus（Linnaeus））变应原进行分离,鉴定其主要及次要变应原,采用蛋白纯化技术获取梭子蟹天然的主要变应原并进行鉴定,为标准化变应原疫苗的研制提供理论依据.方法：取常规方法制备梭子蟹浸出液,经SDS-PAGE分离,测定各组分的相对分子量;同时用26例对蟹过敏的病人血清进行Western blot,鉴定其主要及次要变应原;利用快速制备液相色谱（FPLC）纯化技术（凝胶过滤层析和离子交换层析）获取主要变应原并作鉴定.结果：SDS-PAGE显示梭子蟹有19条可辨蛋白带,分子量在13 000～90 000之间,其中主带有9条,分子量是20 900、24 200、27 100、29 200、33 700、38 900、48 700、74 700、89 100;Western blot结果表明,26例蟹过敏患者血清全部呈阳性反应,浸出液中共有5条致敏条带,其中分子量在74 400、48 700的是主要变应原,阳性反应率均为100%;纯化后获取了74400、48700的主要变应原;经过免疫鉴定证实其具有免疫活性.结论：梭子蟹74 400和48 700的变应原为主要变应原,层析技术可以对分子量为74400和48700的主要变应原成分进行纯化.     

Isolation and identification of pollen allergens of Artemisia scoparia

The allergenic proteins of Artemisia scoparia pollen were separated and identified with ammonium sulfate precipitation, ion-exchange chromatography, gel filtration, and RAST-inhibition techniques. The important allergenic component Artemisia VI b that constitutes 29% of total protein in the extract was purified to homogeneity. It was found to be an acidic protein with isoelectric point 3.8 and molecular weight of 14,300. It was rich in carbohydrate, but the carbohydrate portion did not appear to be important for allergenicity. In the crossed immunoelectrophoresis reference pattern of the whole pollen extract, 37 precipitin lines could be identified on the anodic side, whereas Artemisia VI b could be observed as a single precipitin line. Immunologically, the whole pollen extract of A. scoparia demonstrated shared antigenic and allergenic determinants with Ageratum conyzoides-pollen extract. The use of fast protein liquid chromatography in partial purification of allergenic components is also discussed.     

Molecular cloning and characterization of hazel pollen protein (70 kD) as a luminal binding protein (BiP): a novel cross-reactive plant allergen

BACKGROUND: Tree pollen contains many allergens showing cross-reactivity to proteins from pollen, seeds, and fruits of different plant species. Amongst Fagales, responsible for several allergenic responses, hazel provides the best material to study pollen as well as food allergens in one species. The aim of this study was to identify and characterize the physiological function of an allergen from hazel pollen and to determine possible cross-reactivity to proteins from hazelnut. METHODS: Monoclonal antibodies (mAbs) against hazel pollen crude extract were produced. On the basis of IgE binding, demonstrated by sera from patients allergic to hazel pollen, one mAb indicating the best correlation has been selected, and the putative allergen was purified by preparative gel electrophoresis. Isoforms were investigated by two-dimensional PAGE, and for molecular identification a hazel pollen cDNA library was constructed. In situ localization of the allergen during pollen development was performed by immunofluorescence labelling. RESULTS: Immunological staining of crude hazel pollen extract with specific IgE and mAb revealed a 70-kD protein. Immunoblot studies with mAb showed cross-reactive proteins of 70-72 kD in different plant tissues and species. After protein purification, the IgE-binding reactivity of the allergen has been reconfirmed, and two isoforms were detected. Molecular cloning identified the allergen as a luminal binding protein (BiP) of the Hsp70 family with 88-92% sequence identity in various plants. Further immunocytological studies indicated involvement of BiP during pollen development. CONCLUSIONS: Chaperons like BiP play an important role in protein synthesis and in the protection of cellular structures during stress-related processes. Because of their highly conserved protein sequences, we propose that such allergens could be responsible for at least a part of the allergenic cross-reactivity between proteins from different pollens and plant foods.     

Isolation, expression and immunological characterization of a calcium-binding protein from Parietaria pollen

The diagnosis and therapy of allergic disorders are usually performed with crude extracts which are a heterogeneous mixture of proteins with different allergenic potency. The knowledge of the allergenic composition is a key step for diagnostic and therapeutic options. Parietaria judaica pollen represents one of the main sources of allergens in the Mediterranean area and its major allergens have already been identified (Par j 1 and Par j 2). In addition, inhibition studies performed using a calcium-binding protein (CBP) from grass pollen (Phl p 7) showed the presence of a homologue of this cross-reactive allergen in the Parietaria extract. Screening of a cDNA library allowed us to isolate a 480bp cDNA containing the information for an 87 AA long protein with high level of homology to calcium-binding proteins from other allergenic sources. It was expressed as a recombinant allergen in Escherichia coli and purified by affinity chromatography. Its expression allowed us to study the prevalence of this allergen in a population of allergic patients in southern Europe. Immunoblotting and inhibition studies showed that this allergen shares a pattern of IgE epitopes in common with other 2-EF-hand calcium-binding proteins from botanically non-related species. The immunological properties of the Pj CBP were investigated by CD63 activation assay and CFDA-SE staining. In conclusion, DNA recombinant technology allowed the isolation, expression and immunological characterization of a cross-reactive calcium-binding protein allergen from Parietaria judaica pollen.     

The influence of a residual group in low-molecular-weight allergoids of Artemisia vulgaris pollen on their allergenicity,IgE- and IgG-binding properties

BACKGROUND: Reaction of epsilon-amino groups of lysine with potassium cyanate, maleic, or succinic anhydride leads to allergoids of low molecular weight. No study has been performed to compare their properties and investigate the influence of a residual group on allergenicity and human IgE- and IgG-binding of these derivatives. METHODS: Allergoids of a pollen extract of Artemisia vulgaris were obtained by means of potassium cyanate, and succinic and maleic anhydride. Biochemical properties were investigated by determination of amino groups, enzyme activity, isoelectric focusing IEF and SDS-PAGE. IgE- and IgG-binding was determined using immunoblots and ELISA inhibition. Allergenicity was investigated by skin prick tests (SPT) on a group of 52 patients, of which 6 were control subjects, 30 were patients with no previous immunotherapy (IT), and 16 were patients undergoing immunotherapy. RESULTS: The same degree of amino-group modification (more than 85%), residual enzyme activity (less then 15%), IEF, and SDS-PAGE pattern were noted. In the immunoblots of IgE-binding, there was more pronounced reduction in the succinyl and maleyl derivatives than in the carbamyl one. IgG-binding was less affected by carbamylation than by acid anhydride modification. The SPT showed that the succinylated derivative had the most reduced allergenicity (98% showed a reduced wheal diameter when tested with the succinyl derivative, 87% with the maleyl allergoid, and 83% with the carbamyl allergoid). The most significant difference among allergoids could be seen in the group of patients with high skin reactivity (83% of patients showed no reaction to the succinyl derivative when compared to the value of 28% for the carbamyl derivative or 22% for the maleyl derivative). CONCLUSIONS: According to our results, all three modification procedures yielded allergoids with a similar extent of modification. No single biochemical parameter investigated in the study could predict the degree of reduced allergenicity in vivo. The most reduced allergenicity was seen in the succinyl derivative while the preservation of IgG binding epitopes was of the highest degree for the carbamyl derivative.     

Comparison of allergenicity and immunogenicity of an intact allergen vaccine and commercially available allergoid products for birch pollen immunotherapy

BACKGROUND: Specific immunotherapy with intact allergen vaccine is a well-documented treatment for allergic diseases. Different vaccine formulations are currently commercially available, the active ingredient either being intact allergens or chemically modified allergoids. The rationale behind allergoids is to decrease allergenicity while maintaining immunogenicity. However, data from the German health authorities based on reporting of adverse events over a 10-year period did not indicate increased safety of allergoids over intact allergens. OBJECTIVE: The objective of this study was to investigate the effect of chemical modification on allergenicity and immunogenicity comparing four commercial allergoid products for birch pollen immunotherapy with an intact allergen vaccine. METHODS: Solid-phase IgE inhibition and histamine release assays were selected as model systems for allergenicity, and a combination of human T cell proliferation and IgG titres following mouse immunizations were used to address the immunogenicity of the intact allergen vaccine and the four allergoids. In all assays, the products were normalized with respect to the manufacturer's recommended maintenance dose. RESULTS: IgE inhibition experiments showed a change in epitope composition comparing intact allergen vaccine with allergoid. One allergoid product induced enhanced histamine release compared to the intact allergens, while the other three allergoids showed reduced release. Standard T cell stimulation assays using lines from allergic patients showed a reduced response for all allergoids compared with the intact allergen vaccine regardless of the cell type used for antigen presentation. All allergoids showed reduced capacity to induce allergen-specific IgG responses in mice. CONCLUSION: While some allergoids were associated with reduced allergenicity, a clear reduction in immunogenicity was observed for all allergoid products compared with the intact allergen vaccine, and the commercial allergoids tested therefore do not fulfil the allergoid concept.     

Identification and characterization of the allergenic proteins of Bahia grass (Paspalum notatum) pollen

BACKGROUND: Pollen of Bahia grass (Paspalum notatum) represents a major cause of type I allergy in diverse geographical areas, particularly in the southeastern coastal plain area of the United States. The aqueous protein extract of Bahia grass pollen contains the allergenically active components that produce skin-test-positive reactions in sensitive patients. OBJECTIVE: The emphasis of this study included the identification and characterization of the allergenic proteins present in the crude protein aqueous extract of Bahia grass pollen. METHODS: The crude extract of Bahia grass pollen, partially purified by isoelectric focusing and fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was electroblotted onto nitrocellulose membranes, probed with sera from patients skin test positive to Bahia grass and detected using anti-human IgE conjugated peroxidase. RESULTS: Four allergenic proteins of Bahia grass pollen with estimated molecular weights of 45, 33, 31 and 28 kD were identified and characterized. Following treatments with deglycosylation enzymes, the 4 allergens retained their antigenic reactivity with Bahia-grass-allergic patient sera containing polyclonal IgE antibodies. CONCLUSION: The crude extract of Bahia grass pollen contains many proteins but only 4 have allergenic reactivity. Following deglycosylation treatment, Bahia grass allergenic proteins have retained their antigenic reactivity with Bahia-grass-allergic patient sera containing polyclonal IgE antibodies. Four proteins reactive with IgE were detected, but the 33-kD protein (pI of 6.59) was the most reactive.     

SDS-PAGE analysis of M. leprae protein antigens reacting with antibodies from sera from lepromatous patients and infected armadillos.

Studies have been conducted to characterize M. leprae bacilli derived from infected armadillos. First, the proteins of the mycobacterial extracts were fractionated by SDS-PAGE. Subsequently, the proteins in the gel were electrophoretically transferred on a strip of nitrocellulose paper by the technique of 'electrophoretic blotting'. The separated bacterial protein bands, thus immobilized on the nitrocellulose paper were made to react immunologically with sera from the lepromatous patients, infected armadillo sera and other experimental mycobacterial antisera. It was observed that a majority of M. leprae proteins contained antigenic determinants also present on proteins of BCG. In addition, only two specific antigen bands of 33KD and 12KD were conspicuously detected by the patients' sera and the infected armadillo sera. These substances were further identified as polysaccharides or glycoproteins since they could only be stained by Schiff's reagent or alcian blue. Only 12KD glycoprotein band reacted with concanavalin A, whereas wheat germ agglutinin (WGA) did not show any reaction with them. These 33KD and 12KD glycoprotein antigens were found to lose their antigenicity after pepsin treatment and can be considered as glycoproteins. Further, radiolabelling experiments showed that 12KD antigen underwent radioiodination under usual conditions, but 33KD glycoprotein failed to be similarly radiolabelled. It is suggested that these protein antigens have M. leprae specific determinants on a cross-reacting component.     

Purification and physicochemical characterization of Schistosoma mansoni egg allergen recognized by mouse sera obtained at an acute stage of infection

A major allergenic component recognized by mouse sera obtained at an acute stage of infection was purified from soluble egg antigen preparation (SEA) of Schistosoma mansoni by anion-exchange chromatography on DE52 and gel chromatography on Sephadex G-150. The purified allergen showed homogeneity by immunoelectrophoresis and by polyacrylamide gel electrophoresis. Its apparent molecular weight was 210,000 by gel chromatography on Sephadex G-200. This purified allergen could bind to Con A-Sepharose 4B, indicating its glycoprotein nature. After amino acid analysis, aspartic acid, glutamic acid, serine and theonine were found as the major amino acids. The allergenic activity was destroyed by heating at 100 degrees C for 60 min and by pronase or periodate treatment. By double diffusion in agar gel, this purified allergen gave a strong single band against acute (8 w) stage serum, which fused to the major band formed by crude SEA. On the other hand, it showed a very faint band against chronic (22 w) stage serum, which is apparently different from the main band formed between crude SEA and the chronic stage serum. When specific IgE or IgG antibody titers in the serum of human schistosomiasis mansoni cases were measured by ELISA using this purified allergen, the results showed good correlation with those obtained by using crude SEA. Thus, this purified allergen is not only a major allergen in the acute stage of murine schistosomiasis but also an allergen in human schistosomiasis mansoni.     

Reactivity of T cells with grass pollen allergen extract and allergoid.

BACKGROUND: Successful allergen-specific immunotherapy is achieved with progressively increasing doses of allergen or allergoid. In order to gain further insight into the mechanism of action of allergoids several in vitro investigations were conducted. METHODS: Peripheral blood mononuclear cells (PBMC) from grass pollen allergic and nonallergic subjects were stimulated with either grass pollen extract or allergoid and the proliferation and cytokine production (IL-5, IFN-gamma) were measured. Similar investigations were performed with Phl p 5-specific T cell lines (TCL) and clones (TCC). Dendritic cells and PBMC were compared in terms of their relative efficacies as antigen-presenting cells. RESULTS: Both allergen and allergoid induced proliferation and Th2 and Th1 cytokine synthesis by PBMC of allergic subjects, whereas PBMC of nonallergic subjects did not produce IL-5. The maximum level of IL-5 was obtained with a lower concentration than was necessary for maximal IFN-gamma production. Higher stimulation doses of allergen and allergoid shifted the cytokine profiles towards a Th1 phenotype. TCL and TCC clearly showed reactivity with both allergen and allergoid when using autologous PBMC for antigen presentation, but compared with the native allergen the reactivity of the allergoid was reduced with most of the TCC. Using dendritic cells for antigen presentation a pronounced increase of stimulation of the TCC especially for the allergoids becomes obvious. CONCLUSION: In common with grass pollen allergen the corresponding allergoids possess a strong allergen-specific T cell-stimulating capacity. However, the degree of T cell stimulation by the allergoid seems to be dependent on the type of the antigen-presenting cell. Both, allergen and allergoid, can modulate T cell responses in a dose-dependent manner.     

Analysis of IgE binding proteins of mesquite (Prosopis juliflora) pollen and cross-reactivity with predominant tree pollens

Pollen from the mesquite tree, Prosopis juliflora, is an important source of respiratory allergy in tropical countries. Our aim was to partially characterize the IgE binding proteins of P. juliflora pollen extract and study cross-reactivity with prevalent tree pollen allergens. Intradermal tests with P. juliflora and five other tree pollen extracts were performed on respiratory allergy patients from Bikaner (arid) and Delhi (semi arid). Prosopis extract elicited positive skin reactions in 71/220 of the patients. Sera were collected from 38 of these 71 patients and all demonstrated elevated specific IgE to P. juliflora. Immunoblotting with pooled patients' sera demonstrated 16 IgE binding components, with components of 24, 26, 29, 31, 35, 52, 58, 66 and 95 kDa recognized by more than 80% of individual patients' sera. P. juliflora extract is allergenically potent requiring 73 ng of self-protein for 50% inhibition of IgE binding in ELISA inhibition. Cross-inhibition assays showed close relationship among P. juliflora, Ailanthus excelsa, Cassia siamea and Salvadora persica. IgE binding components of 14, 41, 52 and 66 kDa were shared allergens whereas 26 and 29 kDa were specific to P. juliflora. The findings suggest that purification of cross-reactive allergens will be helpful for diagnosis and immunotherapy of tree pollen allergic patients.     

Physicochemical and immunologic characterization of low-molecular-weight allergoids of Dactylis glomerata pollen proteins

BACKGROUND: Orchard grass (Dactylis glomerata) pollen proteins were chemically modified by means of acid anhydrides (maleic and succinic anhydride) to obtain low-molecular-weight allergoids. Chemical modification in both cases led to the replacement of one positive charge (epsilon amino group of Lys) by one negative charge, yielding proteins with changed physicochemical properties in comparison to the native orchard grass-pollen proteins. METHODS: Physicochemical characterization of derivatives was done by gel chromatography, SDS-PAGE, and isoelectric focusing. To examine the IgE-binding properties of these derivatives, we carried out immunoblotting. To examine the ability of derivatives to induce IgG production, we immunized rabbits. Skin prick testing with the allergoids was performed on 15 individuals allergic to orchard grass pollens and on two healthy subjects. RESULTS: It was shown that the modified proteins retain their original molecular weights, but change pI to more acidic values. In the case of allergoids, a strong reduction in IgE binding was found. Immunization of rabbits with allergoids showed that the derivatives retain the ability to induce IgG production, and that the antisera obtained in such a way react to native (unmodified) extract. The ability of derivatives to induce allergic reaction was significantly reduced. The patients (86.6%) included in our study exhibited less than 50% of native extract response. Among them, 53.3% had no response to one or both allergoids. CONCLUSIONS: These modification procedures yield allergoids with a reduced allergenic activity and preserved immunogenic potential suitable for use in immunotherapy.     

Identification of a plane pollen lipid transfer protein (Pla a 3) and its immunological relation to the peach lipid-transfer protein, Pru p 3

BACKGROUND: An association between plane tree pollen allergy and plant food allergy has been described, but the cross-reacting allergens have not yet been identified. The aim of this study was the identification of homologous non-specific lipid-transfer proteins (nsLTPs) in plane pollen, and to investigate its immunological relationship with the peach LTP, Pru p 3. METHODS: Three different patient groups were recruited in Spain: 22 plane pollen-allergic patients without food allergy (A), 36 plane pollen-allergic patients with peach allergy (B) and 10 peach-allergic patients without plane pollen allergy (C). Proteins from plane pollen extract were fractionated by ion-exchange and reversed-phase chromatography. Further methods applied were N-terminal amino acid sequence analysis, immunoblotting, enzyme allergosorbent test, CAP and basophil histamine release assays. RESULTS: A 10 kDa IgE-reactive protein was purified from plane pollen and identified as nsLTP. Pla a 3 was characterized as a minor allergen (27.3%) in plane pollen-allergic patients without food allergy (A) and as a major allergen in plane pollen-allergic patients with peach allergy (B) showing a prevalence of IgE-reactivity of 63.8%. Group B contained patients sensitized to Pru p 3 without IgE-reactivity to plane-LTP (16.6%). By contrast, Pla a 3 IgE-reactive patients without sensitization to Pru p 3 could be found (16.6%). The sera of patients sensitized to both LTPs (50%), Pla a 3 and Pru p 3, showed different biological activity in histamine release assay: depending on individual patient's sera tested, Pla a 3 showed a similar, a stronger or a weaker allergenic potency in comparison with Pru p 3. CONCLUSIONS: Plane LTP is a major allergen in plane pollen-allergic patients with peach allergy recruited in the Mediterranean area. The results of histamine release tests and different IgE-binding profiles pointed towards the existence of species-specific IgE epitopes. Likewise, no general conclusion on the sensitizer could be made.     

Measurement of basophil-activating capacity of grass pollen allergens, allergoids and hypoallergenic recombinant derivatives by flow cytometry using anti-CD203c

BACKGROUND: The assessment of the basophil-activating potential is an important aspect in the development of improved preparations for specific immunotherapy. The aim of the study was to evaluate the suitability of CD203c expression as a measure of basophil activation to compare allergoids with original allergen extracts, and recombinant hypoallergenic allergen derivatives with recombinant wild-type and natural allergens. METHODS: Heparinized whole blood samples from grass pollen allergic subjects were stimulated with grass pollen allergens and allergen derivatives followed by labelling of the basophils with PE-conjugated anti-CD203c. After lysis of the erythrocytes and fixation, the basophils were detected by flow cytometry. In some experiments, histamine release was determined simultaneously. RESULTS: Grass pollen allergoids revealed a 10-10 000-fold reduction of basophil-activating capacity measured by CD203c expression. The deletion mutant DM4 of rPhl p 5b showed stronger hypoallergenic characteristics in a range of 50-10 000-fold reduction, whereas a combination mutant of rPhl p 5b and Phl p 6 revealed less hypoallergenic features. Histamine release experiments led to a similar outcome as CD203c measurement. CONCLUSIONS: The measurement of CD203c expression on basophils by flow cytometry provides a rapid and sensitive method for the estimation of the allergic or hypoallergenic features of allergen preparations. The results demonstrated the hypoallergenicity of grass pollen allergoids and of the rPhl p 5b variant DM4, which may be a candidate in future preparations for specific immunotherapy.