Ubiquitous structures responsible for IgE cross-reactivity between tomato fruit and grass pollen allergens

The simultaneous presence of IgE reactivity to tomato fruit and grass pollen allergens is evident in many patients with allergy and may be caused by cross-reactivity. Using sera from polysensitized patients with a positive enzyme allergosorbent test (EAST) result (score > 2), we tested reactivity to both allergen sources. IgE reactivity against both extracts was demonstrated in eight serum samples, and cross-reactivity was confirmed by the EAST inhibition assay. The structures responsible for this cross-reactivity were identified by Western blotting: five of the eight sera demonstrated a 16 kd protein in both extracts, which was identified as profilin. Additionally, seven of the eight sera showed IgE binding to epitopes on carbohydrate moieties, which contained alpha 1, 3 fucosylations. To determine the allergens of tomato fruit extract, we performed two-dimensional polyacrylamide gel electrophoresis blotting. We were able to demonstrate one highly concentrated and about 20 weaker proteins possessing terminal fucose residues. These are similarly found in grass pollen extracts. It is therefore postulated that the cross-reactivity is affected by profilins and similar carbohydrate determinants. If carbohydrate structures can provoke IgE cross-reactivity between phylogenetically distant species, such structures may play an important role in sensitization and mediator release. The ubiquitous nature of the IgE-binding determinants was studied by additional EAST inhibition tests with tomato allergen disks and extract from birch pollen, mugwort pollen, apple, and celery, leading to significant inhibitions among all these allergen sources. Epitopes exclusive to grass pollen and tomato have not been detected.     

Modified intra-arterial calcium stimulation with venous sampling test for preoperative localization of insulinomas

BACKGROUND: Both genetic and environmental factors are thought to contribute to specific IgE responses, however, the relative contribution of each in the responses to individual ryegrass pollen allergens is largely unknown even though some responses to allergens have been linked to certain HLA complexes. OBJECTIVE: Using a large group of monozygotic and dizygotic twins, this study was designed to investigate the IgE binding profiles of individual ryegrass pollen (Lolium perenne) components to assess the relative contribution of genetic and environmental factors in determining IgE responses to specific allergens. METHODS: Ryegrass pollen proteins were separated by electrophoresis and immunoblotted with sera from 191 pairs of twins where at least one sibling had a SPT > 2 mm to perennial ryegrass. Concordance levels for individual ryegrass pollen components were compared between monozygotic and dizygotic twins in a subset group where both twins had SPT > 3 mm to perennial ryegrass. RESULTS: Immunoblotting revealed 23 individual IgE-binding components from ryegrass pollen. Although there was a significantly greater proportion of monozygotic twins with SPT wheals greater than 3 mm when compared with the dizygotic twins, the mean case-wise concordance for the immunoblot components was similar for both groups of twins. The mean case-wise concordance when at least four pairs of sera were involved was 44% for the MZ twins (n=11 components) and 45% for the DZ twins (n=12 components). We found no significant differences in concordance levels for any of the 23 individual components including allergens previously associated with HLA. CONCLUSION: Evidence for genetic control of allergen-specific IgE responses in a large population sample of twins to individual ryegrass allergens is limited, indicating that the IgE responses to specific ryegrass pollen allergens are determined largely by environmental factors.     

Effects of IL-4 and IL-13 on total and allergen specific IgE production by cultured PBMC from allergic patients determined with recombinant pollen allergens

BACKGROUND: Interleukin (IL)-4 and IL-13 have been shown to be potent switch factors for IgE synthesis in human B cells. OBJECTIVE: In this study we investigated the effects of recombinant human IL-4 and IL-13 on total and allergen specific IgE synthesis by peripheral blood mononuclear cells (PBMC) from pollen allergic patients and healthy control individuals. METHODS: Peripheral blood mononuclear cells (PBMC) from allergic patients were investigated for their capacity to produce allergen specific IgE in vitro. Total protein extracts from birch pollen and timothy grass pollen as well as purified recombinant birch pollen allergens, Bet v I, birch profilin (Bet v II) and recombinant timothy grass pollen allergens, Phl p I, Phl p II, and Phl p V were used to measure specific IgE-antibody synthesis in cell culture supernatants by IgE-immunoblot and ELISA. RESULTS: PBMC obtained from allergic patients spontaneously secreted allergen specific IgE in the culture supernatants. Addition of Interleukin 4, Interleukin 13 and anti-CD40 antibody to the cultures alone or in combinations significantly induced total IgE production whereas allergen specific IgE production was not affected. CONCLUSION: Our results indicate that the peripheral blood of allergic individuals contains long lived allergen specific B cells which have already switched to IgE production and which are not sensitive to IL-4 and IL-13 treatment. These results may have implications on attempts to use cytokines or cytokine antagonists in therapy of Type I allergy.     

Hematuria in the child. Investigation plan in pediatric practice

BACKGROUND: Grass pollen extracts are complex mixtures consisting of different major allergenic and non-allergenic components. Phl p 4 is an important allergen, because more than 75% of grass pollen allergic patients produce specific IgE antibodies against group 4 allergens. OBJECTIVE: This study was designed to investigate the specificity of monoclonal antibodies (MoAbs) produced against Phl p 4 and to verify the presence of group 4-like proteins in different grass pollen. Furthermore the usefulness of MoAbs for quantification of group 4 allergens was studied. METHODS: Group 4 analogues were investigated by immunoblotting and ELISA inhibition using three MoAbs. The specificity of antibodies was studied using isolated group 1 and group 5 allergens. Quantification of group 4 allergen was achieved by a two-site solid-phase ELISA. Phl p 4 was purified from whole pollen extract by chromatographic or electrophoretic techniques and used as standard. RESULTS: The MoAbs studied bound strongly to proteins from timothy grass pollen extract at a mw of 55 kDa and a pI of 9.0-9.3. Phl p 4 homologes with similar mw were detected in Dactylis glomerata, Festuca pratensis, Holcus lanatus, Poa pratensis, Lolium perenne. Epitope mapping showed that all three MoAb recognized unrelated regions on Phl p 4. A two-site binding ELISA using MoAbs was developed for determination of Phl p 4 in Phleum pratense extracts. The method was able to evaluate group 4 in mass units with a working range between 150 and 2000 ng/mL. The absolute amounts of group 4 in extracts of several grasses varied considerably but was always-less than 1% of the total protein. CONCLUSION: Group 4 homologes are present in the various grass extracts but to different extents. The group 4 ELISA could be very useful as a additional tool for providing information concerning the composition of grass pollen extracts.     

Molecular basis of IgE-recognition of Lol p 5, a major allergen of rye-grass pollen

Grass pollen, especially of rye-grass (Lolium perenne). represents an important cause of type I allergy. Identification of IgE-binding (allergenic) epitopes of major grass pollen allergens is essential for understanding the molecular basis of interaction between allergens and human IgE antibodies and therefore facilitates the devising of safer and more effective diagnostic and immunotherapy reagents. The aim of this study was to identify the allergenic epitopes of Lol p 5, a major allergen of rye-grass pollen, immunodissect these epitopes further so that the amino acid residues critical for antibody binding can be determined and investigate the conservation and nature of these epitopes within the context of the natural grass pollen allergens. Peptides, 12-13 amino acid residues long and overlapping each other by 4 amino acid residues, based on the entire deduced amino acid sequence of the coding region of Lol p 5, were synthesised and assayed for IgE-binding. Two strong IgE-binding epitopes (Lol p 5 (49-60) and (265-276), referred to as peptides 7 and 34, respectively) were identified. These epitopes were further resolved by truncated peptides and amino acid replacement studies and the amino acid residues critical for IgE-binding determined (Lol p 5 (49-60) residue Lys57 and (265-276) residue Lys275). Sequences of these epitopes were conserved in related allergens and may form the conserved allergenic domains responsible for the cross-reactivity observed between pollen allergens of taxonomically related grasses. Furthermore, due to its strong IgE-reactivity, synthetic peptide Lol p 5 (265-276) was used to affinity-purify specific IgE antibodies which recognised proteins of other clinically important grass pollens. further indicating presence of allergenic cross-reactivity at the level of allergenic epitope. Moreover, Lol p 5 (265 276) demonstrated a strong capacity to inhibit IgE-binding to natural rye-grass pollen proteins highlighting the antibody accessibility to these sequences within the context of the natural allergens. Strong IgE-binding epitopes of Lol p 5 have been identified down to single critical amino acid residues and are shown to occur as linear or continuous domains in the natural conformation of natural Lol p 5 and other group 5 grass pollen allergens. The fact that such an allergenic synthetic epitope has the capacity to strongly inhibit IgE-binding to natural allergens highlight its potential for use as a candidate in future therapeutics to treat pollen-associated allergies.     

Measurement of allergen-specific IgD and correlation with allergen-specific IgE

A new method for the measurement of allergen-specific IgD (as-IgD) was developed by modifying the ImmunoCAP assay (Pharmacia), and amplification of the signal with a goat anti-human/rabbit anti-goat detection system. The assay was sensitive enough to measure as-IgD in serum samples. The specificity of the assay was examined using inhibition tests with excess corresponding and non-corresponding allergens. For the different allergens inhibition rates between 56% (house dust mite) and 88% (cat) could be achieved. Non-corresponding allergens did not inhibit the as-IgD binding. Total IgE and allergen-specific IgE (as-IgE) was measured using the ImmunoCAP system. Total IgD was measured using a sandwich ELISA. As-IgD was measured in serum samples from 51 atopic and 23 non-atopic subjects, and the correlation with as-IgE was examined. As-IgD was detected in both atopics and non-atopics but at higher levels in atopics. As-IgD against birch pollen and timothy pollen allergen was found to be increased in atopics with IgE directed against these allergens compared to atopics without IgE against these allergens (P < 0.02 and P < 0.03). As-IgD against birch pollen allergen was higher in atopics with IgE specific to this allergen than in non-atopics (P < 0.02). In contrast to total IgE and total IgD, significant correlations were observed between as-IgD and as-IgE against timothy pollen (r = 0.34, P < 0.04), birch pollen (r = 0.38, P < 0.05) and cat dander allergen (r = 0.52, P < 0.01). The observed correlations between as-IgD and IgE suggest that IgD and IgE may be similarly regulated, and thus the measurement of as-IgD may give further insight into the regulation of IgE.     

Human IgG monoclonal antibodies that modulate the binding of specific IgE to birch pollen Bet v 1

Birch pollen allergy is a very frequent pathology in Europe and North America. More than 95% of the tree pollen allergic patients display IgE reactivity against Bet v 1, the major birch pollen allergen. Starting with PBL from a patient desensitized by immunotherapy, we have generated five B cell lines (BAB1 to BAB5) that secrete human IgG mAbs of high affinity for Bet v 1. Although competition studies indicated that these human IgG mAb recognized different epitopes, broad cross-reactivity was found with Bet v 1 homologous allergens present in tree pollens and plant-derived foods. When tested for interference with allergic patients' IgE, BAB1 inhibited (by 80-100%) the binding of IgE to nitrocellulose-blotted Bet v 1, while BAB2 enhanced it. The biologic significance of the ability of BAB1 to interfere with patients' IgE binding is indicated by the finding that BAB1 completely inhibited Bet v 1-induced histamine release from allergic patients' basophils. Allergen-specific human IgG mAbs such as BAB1, which presents high blocking activity in both immunochemical and cellular IgE competition experiments, might have therapeutical application.     

Race and clinical outcome in patients with carcinoma of the uterine cervix treated with radiation therapy

Epidemiologic and in vitro data have shown that the association of house-dust mite (HDM) allergy and snail allergy in the same patients was due to cross-reactivity between HDM and snail allergenic components. However, the cross-reacting allergen(s) have not yet been identified. In vitro reactivity of seven patients' sera to the various extracts and hemolymph of four different Helix snail species was analyzed by IgE detection and immunodots and Western blots. Cross-reactivity between snails and Dermatophagoides pteronyssinus was assessed by immunodot and ELISA inhibition in two patients. Heterologous inhibition of the snail immunodot and ELISA was observed in one serum. Western blotting showed a specific binding on all four snail species extracts; molecular weights of snail allergens ranged from < 21 to 200 kDa. Marked individual differences were observed in the seven sera under study; most sera demonstrated IgE recognition of multiple bands, illustrating that no single allergen is responsible for cross-reactivity between snail and mite. These results confirm that cross-reactivity exists between snails of the Helix genus and HDM. This cross-reactivity, involving more than a single allergen, may be of clinical significance in atopic patients allergic to D. pteronyssinus. The identity of the cross-reacting allergens remains to be determined. Potential candidates include the thermostable minor allergens of D. pteronyssinus, tropomyosin and hemocyanin.     

Immunological and biological properties of Bet v 4, a novel birch pollen allergen with two EF-hand calcium-binding domains

We have isolated a cDNA clone coding for a birch pollen allergen, Bet v 4. The deduced amino acid sequence of Bet v 4 contained two typical EF-hand calcium-binding domains. Sequence similarities of Bet v 4 to calmodulin are primarily confined to the calcium-binding domains. However, significant sequence similarities extending outside the Ca2+-binding sites were found with a recently described group of pollen-specific allergens of Brassica and Bermuda grass. Both EF-hand domains of Bet v 4 are able to bind Ca2+, as demonstrated by 45Ca2+ blot overlay of wild type and calcium-binding deficient mutants of Bet v 4. Among pollen-allergic patients, protein-bound Ca2+ was not an absolute requirement for IgE recognition of Bet v 4. However, disruption of the carboxyl-terminal Ca2+-binding domain indicated that most IgE antibodies from allergic patients are directed against this site. IgE inhibition experiments suggested that Bet v 4 represents a highly cross-reactive pollen allergen. Pre-absorption of allergic sera with Bet v 4 drastically reduced IgE binding to proteins of similar molecular weight in pollen extracts from distantly related plant species (e.g. timothy grass, mugwort, lily) but not in extracts from plant-derived foodstuff. To test for a possible biological role in pollen germination and tube growth, we introduced recombinant Bet v 4 protein into growing lily pollen tubes by iontophoresis. As a result, cytoplasmic streaming stopped in the vicinity of the electrode tip, and a slight depolarization of the membrane voltage was measured. These effects were not observed with Ca2+-binding deficient mutants of Bet v 4. Thus, Bet v 4 and homologous proteins represent a new class of pollen-specific Ca2+-binding allergens that may have a physiological role as inhibitors of cytoplasmic streaming in outgrowing pollen tubes.     

In vitro cultivation of Wolbachia pipientis in an Aedes albopictus cell line

Bet v 1, the single major allergen from birch pollen, shares IgE epitopes with all major tree pollen allergens from closely related species such as alder, hazel, hornbeam, beech, and European chestnut. Because of high sequence homologies among these allergens and the well-studied cross-reactivities on B cell epitopes, Bet v 1 is a representative model protein which can be used for in vitro studies. The cDNA coding for Bet v 1, the single major allergen from birch pollen, was cloned into the T7-based Escherichia coli expression system pMW 175/BL21(DE3) and synthesized as a nonfusion protein. In contrast to other E. coli systems (e.g., pKK233-2/JM105), this system produces high levels of readily extractable proteins corresponding to 5-10% of E. coli total protein, the percentage varying with culture conditions. The overall yield was 8-10 mg of purified recombinant protein per liter of culture medium. The recombinant allergen was purified by several steps, including ion-exchange and hydrophobic interaction chromatography. The purified recombinant allergen showed identical immunological properties with the respective natural counterpart. The use of recombinant allergens of high purity is expected to result in more accurate diagnostic procedures, but possibly also in a superior immunotherapy of Type I allergic diseases when compared with methods using crude allergen extracts containing various amounts of allergen concentrations.     

Evaluation of allergenic potency by REAST inhibition. A new tool for the standardization of allergenic extracts

The potency of allergenic extracts can be determined in vitro by RAST inhibition, and this has become the preferred method for the standardization of allergens. A disadvantage of this technique is the impossibility of obtaining data about allergens bound to the solid phase, i.e., the counterpart of the inhibiting extract. The REAST (reverse enzyme allergosorbent test) is based on the capture of IgE by a specific antibody bound to microtiter wells, the reaction of captured IgE with biotinylated allergen and the development of a colour reaction by subsequent addition of streptavidin-peroxidase and chromogenic substrate. The addition of an allergen extract in a dose-response fashion competes with the biotinylated allergen and inhibits the test. In the present study REAST inhibition has been evaluated with Dermatophagoides pteronyssinus, Parietaria judaica and mixed grass pollen extracts. The correlation of REAST inhibition with RAST inhibition and both intra-assay and inter-assay reproducibility have been evaluated. REAST inhibition is a potentially valuable new tool for the standardization of allergenic extracts.     

Investigation of different recombinant isoforms of grass group-V allergens (timothy grass pollen) isolated by low-stringency cDNA hybridization--antibody binding capacity and allergenic activity

A cDNA library of timothy grass pollen was screened for homologous isoforms of major group-V allergens by low stringency hybridization with a Phl p 5 (Phleum pratense) probe. After restriction analysis of the 40 clones obtained, 17 were selected for cDNA sequencing. Of these clones, two were unrelated to group-V allergens, six showed high similarity but an incomplete open reading frame and nine had high similarity with a complete open reading frame. Comparison of deduced amino acids of ten complete cDNA clones confirmed the presence of two major isoforms, a and b. Within these two subgroups, only minor sequence variations were observed. Eight isoforms were expressed in Escherichia coli K12 and purified to homogeneity. Although the subgroups a and b could be distinguished by their molecular masses and by binding constants towards monoclonal antibodies, all isoforms turned out to be biochemically similar. Ribonuclease activity as a marker for the biological function of group-V allergens was shown to be in the same range for both subgroups. Analysis of allergenic B-cell responses towards the isoforms in 26 grass pollen allergic patients revealed that the IgE reactivities to the different isoforms were identical for each individual. IgE reactivities and allergenic activities of three isovariants and an allergen of a different group were compared in a selected group of four grass pollen allergic patients by immunoblot, histamine-release and skin-prick tests. The IgE reactivity does not necessarily mirror the allergenic activity of the single molecule, and the variability of allergenic activity between the isovariants does not, in every case, depend on the structural differences of these allergens. We conclude that group-V isoallergens in grass pollen, although they can be structurally different, induce a similar B-cell response but can show variable allergenic activity. Thus, the most allergenic isoform of each important group of allergens should be sufficient for the diagnosis of type-I allergy. Whether the isoallergenic variation has any significant influence on the outcome of immunotherapy in allergic disease still has to be elucidated.     

Immunization with purified natural and recombinant allergens induces mouse IgG1 antibodies that recognize similar epitopes as human IgE and inhibit the human IgE-allergen interaction and allergen-induced basophil degranulation

Molecular characterization of allergens by recombinant DNA technology has made rapid progress in the recent few years. In the present study we immunized mice with aluminum hydroxide-adsorbed purified recombinant major timothy grass pollen allergens (rPhl p 1, rPhl p 2, rPhl p 5), dog albumin, a major animal dander allergen, and proteins with low (beta-lactoglobulin) or no (ribulose diphosphate carboxylase) allergenic potential in humans. Allergens that bind high levels of IgE in humans (Phl p 1, Phl p 5, dog albumin) induced high IgE and IgG1 levels in mice, whereas proteins with little or no allergenic activity in humans failed to induce significant IgE and IgG1 levels in mice. Continuous immunization for a period of 27 wk resulted in the production of mouse IgG1 Abs that recognized recombinant allergen fragments/epitopes defined by IgE Abs of allergic patients. As a consequence, allergen-specific mouse Abs strongly inhibited human IgE binding to the allergens and suppressed the allergen-induced histamine release from human basophils. In summary, our data indicate that 1) the allergenic potency of a protein may be related to its overall immunogenicity and 2) prolonged immunization with single purified recombinant allergens induces protective IgG Abs. The presented experimental in vivo/in vitro system allows the evaluation of Ag preparations (e.g., recombinant allergens) to be used for immunotherapy in humans.     

Non-specific airway hyperresponsiveness in mono-sensitive Sicilian patients with allergic rhinitis. Its relationship to total serum IgE levels and blood eosinophils during and out of the pollen season

BACKGROUND: Initial attempts to evaluate the association between allergic rhinitis and non-specific bronchial responsiveness has produced conflicting results. In fact, some studies showed a strong correlation and other failed to find an association. However, little is known about the effect of natural specific allergen exposure on the bronchial reactivity of mono-sensitive patients with rhinitis in the southern Mediterranean area, in relation to skin reactivity to allergens, total serum IgE levels and blood eosinophils. OBJECTIVES: The significance of the association between allergic rhinitis, and abnormal airway responsiveness with regard to the pathogenesis of asthma is unclear. For this reason, we have studied non-specific bronchial hyperreactivity, in patients with seasonal allergic rhinitis, with reference to the responsible allergen. The aim of the study was to correlate the responsiveness to bronchoprovocation with methacholine in subjects a with allergic rhinitis during and out of the pollen season with total serum IgE and blood eosinophils. METHODS: Fourty-nine non-smoking patients with clinical diagnosis of allergic rhinitis and mono-sensitive skin-prick tests to pollen allergens were enrolled in the study. Twenty patients suffered from seasonal rhinitis to Parietaria pollen, 15 patients to Gramineae pollen and 14 patients to Olea pollen. In all patients lung function measurements (assessed as response to methacholine), total serum IgE and blood eosinophil counts were measured during and out of the pollen season. RESULTS: During pollen season, 16 out of 49 rhinitis patients demonstrated values of bronchial responsiveness measured as response to inhaled methacholine in the asthmatic range whereas out of the pollen season only eight patients were in the asthmatic range. By analysing the results with reference to the responsible allergen, during the pollen season 15 out of 16 patients were Parietaria-sensitive and out of the pollen season seven out of eight patients. Finally, in Parietaria-sensitive rhinitis bronchial responsiveness significantly correlated, during and out of the pollen season, with total serum IgE and with blood eosinophil counts. CONCLUSIONS: Our results are consistent with the hypothesis that Parietaria is more important than Olea and Gramineae as a risk for developing non-specific bronchial hyperresponsiveness. On the whole, present observations provide further evidence that there is an interrelationship of allergen kind, total serum IgE, eosinophil and bronchial hyperresponsiveness suggesting that they may play a role in the development of bronchial asthma in rhinitis patients.     

Probing the molecular basis of allergy. three-dimensional structure of the bovine lipocalin allergen Bos d 2

The three-dimensional structure of the major bovine allergen Bos d 2 has been determined by using x-ray diffraction at 1.8-A resolution. Structurally Bos d 2 is a member of the lipocalin family comprising proteins with transport functions. There is a flat small cavity inside the Bos d 2 protein core suitable for ligand binding, and it is possible that Glu115 and Asn37 inside the core are able to make hydrogen bonds with the ligand. Many allergens from different animals belong to the lipocalin family. The amino acid residue similarities between these lipocalins indicate putative regions for IgE binding. Comparison with the available allergen structures from other sources suggests that these allergens are roughly the same size and that their shape is more spherical than elliptical.     

Allergen sensitization of asthmatic and nonasthmatic schoolchildren in Costa Rica

The prevalence of asthma among schoolchildren in Costa Rica is very high -- at the level of 20-30% -- and the reason is still unknown. A group of children from our previous epidemiologic study was randomly selected in order to establish the relation between asthma symptoms and allergy sensitization to common allergens. Serum samples from children with and without asthma were analyzed for the presence of IgE antibodies to 36 different allergens, for the presence of IgE antibodies to a pool of 10 common allergens, and for total serum IgE. The most prevalent IgE antibodies were those to mite, cockroach, dog, and house-dust allergens with MAST pipettes for the serologic measurements. Positive reactions to house dust, mite, cat, and the two molds (Alternaria and Cladosporium), and food allergens such as egg white, peanut, and shellfish were significantly more prevalent among the asthmatics than the nonasthmatics. Sensitization was equally prevalent at different ages, but the house-dust, mite, cat, dog, cockroach, Alternaria, and egg-white allergens had sensitized boys more often than girls (P < 0.01). The result of the analysis of IgE antibodies to a pool of 10 common allergens by Phadiatop supported the MAST pipette results, showing allergen sensitization in 57.7% of the asthmatic children and 42.3% in the nonasthmatic group. The concentration of IgE was significantly higher among the asthmatic children (372.2 kU/l) than among the nonasthmatic children (249.1 kU/l) (P < 0.00001). Parasitic infestations were not examined in this study, but in most of Costa Rica these have largely been eliminated and could not explain the high total IgE levels. Our data indicate that the very high prevalence of bronchial asthma in Costa Rican schoolchildren can be related to sensitization, especially to airborne indoor allergens such as those of mites, cockroaches, and dogs.     

Value of monoclonal antibody-based assays: advantages and drawbacks

Monoclonal antibodies (mAbs) directed to allergens are highly specific tools in allergen standardization and quantification. Their mono-specificity allows sensitive detection of individual allergens. It is the same quality that asks for caution. MAbs can be too specific: isoforms of allergens can be missed. On the other hand, mono-specific antibodies can also be crossreactive, possibly resulting in overestimation of allergen content. Lower affinity of mAbs compared to IgE may lead to underestimation of the IgE-binding capacity of an allergen extract. For application of mAbs these potential pitfalls should be taken into consideration.     

"Allergen engineering": variants of the timothy grass pollen allergen Phl p 5b with reduced IgE-binding capacity but conserved T cell reactivity

One problem of conventional allergen-specific immunotherapy is the risk of anaphylactic reactions. A new approach to make immunotherapy safer and more efficient might be the application of engineered allergens with reduced IgE-binding capacity but retained T cell reactivity. Using overlapping dodeca-peptides, the dominant T cell epitopes of the timothy grass pollen allergen Phl p 5b were identified. By site-directed mutagenesis outside these regions, point and deletion mutants were generated. Allergen variants were analyzed for IgE-binding capacity with sera of different grass pollen allergic patients by Western blotting, Dot blotting, and EAST inhibition test, and for histamine releasing capacity with peripheral blood basophils from different patients. The deletion mutants revealed significantly reduced IgE reactivity and histamine releasing capacity, compared with the wild-type Phl p 5b. Furthermore, in vivo skin prick tests showed that the deletion mutants had a significantly lower potency to induce cutaneous reactions than the wild-type Phl p 5b. On the other hand, T cell clones and T cell lines from different allergic patients showed comparable proliferation after stimulation with allergen variants and wild-type Phl p 5b. Considering their reduced anaphylactogenic potential together with their conserved T cell reactivity, the engineered allergens could be important tools for efficient and safe allergen-specific immunotherapy.     

Effects of reconstructive surgery for left ventricular anterior aneurysm on ventriculoarterial coupling

Microbial agents are known to play a significant role in aggravating allergic diseases. Recently described viral and bacterial superantigens represent one important strategy by which infectious agents can stimulate the immune response. In previous work, we reported that the staphylococcal toxin toxic shock toxin-1 (TSST-1), a prototypic superantigen, induces in vitro total IgE synthesis after cross-linking T and B cells. This study was carried out to establish a potential link between superantigens and the enhanced IgE response to specific allergens in allergic patients. Peripheral blood mononuclear cells from atopic patients were isolated during and outside the pollen allergen season and stimulated with TSST-1, a prototypic superantigen. Total IgE and interferon-gamma production were measured in supernatants of these cultures. Outside the pollen season, TSST-1 significantly increased total IgE production only in the presence of exogenous interleukin-4, whereas during the pollen season IgE production was significantly enhanced without the need of exogenous interleukin-4. This increase in the absence of exogenous interleukin-4 was associated with significantly lower interferon-gamma production by peripheral blood mononuclear cells stimulated by TSST-1 during the pollen season. Moreover, TSST-1 stimulation of peripheral blood mononuclear cells from inhalant allergic patients was followed by an increased production of allergen-specific IgE that was restricted to the allergen to which the patient was allergic and recently exposed. In addition, TSST-1 induced on B cells the expression of B7.2, a molecule that has recently been demonstrated to enhance T helper 2 responses and to be involved in IgE regulation. This study, by demonstrating that superantigens can augment allergen-specific IgE synthesis and B7.2 expression, provides a mechanism by which microbial superantigens may modulate allergic responses.