Recombinant allergens: the future of the diagnosis and treatment of atopic allergy.
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Allergens↗
Biomedical subjects
D Kraft
Publications and source records attributed to D Kraft.
The immunoglobulin E-allergen interaction: a target for therapy of type I allergic diseases.
The interaction of immunoglobulin E and otherwise harmless antigens (allergens) leads in sensitized individuals through effector cell activation to the immediate induction of a cascade of inflammatory reactions, the hallmark of type I allergy. Recently, the molecular and structural characterization of allergens, specific IgE antibodies and their epitopes has made rapid progress. Here we discuss active and passive strategies for therapy of type I allergy, which are based on interfering with the IgE-allergen interaction.
Allergens↗
Calcium-binding allergens: from plants to man.
Calcium-binding proteins contain a variable number of motifs, termed EF-hands, which consist of two perpendicularly placed alpha-helics and an inter-helical loop forming a single calcium-binding site. Due to their ability to bind and transport calcium as well as to interact with a variety of ligands in a calcium-dependent manner, they fulfill important biological functions in eukaryotic cells. After parvalbumin, a three EF-hand fish allergen, calcium-binding allergens were discovered in pollens of trees. grasses and weeds and, recently, as autoallergens in man. Although only a small percentage of atopic individuals displays IgE reactivity to calcium-binding allergens, these allergens may be important because of their ability to cross-sensitize allergic individuals. Confrontation and stability++ as well as IgE recognition of calcium-binding allergens greatly depend on the presence of protein-bound calcium ions. It is thus likely that hypoallergenic derivatives of calcium-binding allergens can be engineered by recombinant DNA technology for immunotherapy++ of sensitized patients.
Allergens↗
Venoconstrictive thigh cuffs impede fluid shifts during simulated microgravity.
BACKGROUND: This study determined the efficacy of venoconstrictive thigh cuffs, inflated to 50 mmHg, on impeding fluid redistributions during simulated microgravity. METHODS: There were 10 healthy male subjects who were exposed to a 2-h tilt protocol which started in the standing position, and was followed by 30 min supine, 30 min standing, 30 min supine, 30 min of -12 degrees head down tilt (HDT, to simulate microgravity), 15 min of HDT with venoconstrictive thigh cuffs inflated, a further 10 min of HDT, 5 min supine, and 10 min standing. To increase the sensitivity of the techniques in an Earth-based model, 12 degrees HDT was used to simulate microgravity effects on body fluid shifts. Volume changes were measured with anthropometric sleeve plethysmography. RESULTS: Transition to the various tilt positions resulted in concomitant decrements in leg volume (Stand [STD] to Supine [SUP], -3.0%; SUP to HDT, -2.0%). Inflation of the venoconstrictive thigh cuffs to 50 mmHg, during simulated microgravity, resulted in a significant 3.0% increase in leg volume from that seen in HDT (p < 0.01). No significant changes in systemic cardiovascular parameters were noted during cuff inflation. CONCLUSIONS: We conclude that venoconstrictive thigh cuffs, inflated to 50 mmHg for 15 min during 12 degrees HDT, can create a more Earth-like fluid distribution. Cuffs could potentially be used to ameliorate the symptoms of cephalad edema seen with space adaptation syndrome and to potentiate existing fluid volume countermeasure protocols.
Adult↗
Molecular characterization, expression in Escherichia coli, and epitope analysis of a two EF-hand calcium-binding birch pollen allergen, Bet v 4.
Birch pollen belongs to the most potent elicitors of Type I allergic reactions in early spring. Using serum IgE from a birch pollen allergic patient, two cDNA clones (clone 6 and clone 13) were isolated from a birch pollen expression cDNA library constructed in phage lambda gt11. Clone 6 encoded a 9.3 kD two EF-hand calcium-binding protein, designated Bet v 4, with significant end to end sequence homology to EF-hand calcium-binding allergens from weed and grass pollen. Recombinant Bet v 4, expressed as beta-galactosidase fusion protein, reacted with serum IgE from approximately 20% of pollen allergic individuals. Depletion of allergenbound calcium by EGTA treatment lead to a substantial reduction of IgE-binding to Bet v 4, indicating that protein-bound calcium is necessary for the maintenance of IgE-epitopes. The greatly reduced IgE-binding capacity of clone 13, a Bet v 4 fragment that lacked the 16 N-terminal amino acids, indicated that the N-terminus contributes significantly to the proteins IgE-binding capacity. By IgE-inhibition experiments it was demonstrated that recombinant Bet v 4 shared IgE-epitopes with natural Bet v 4 and a homologous timothy grass pollen allergen. Recombinant Bet v 4 may therefore be considered as a relevant crossreactive plant allergen, which may be used for diagnosis and treatment of patients suffering from multivalent plant allergies.
Allergens↗
Conversion of the major birch pollen allergen, Bet v 1, into two nonanaphylactic T cell epitope-containing fragments: candidates for a novel form of specific immunotherapy.
A novel approach to reduce the anaphylactic activity of allergens is suggested. The strategy makes use of the presence of conformational immunoglobulin E (IgE) epitopes on one of the most common allergens. The three dimensional structure of the major birch pollen allergen, Bet v 1, was disrupted by expressing two parts of the Bet v 1 cDNA representing amino acids 1-74 and 75-160 in Escherichia coli. In contrast to the complete recombinant Bet v 1, the fragments showed almost no allergenicity and exhibited random coil conformation as analyzed by circular dichroism. Both nonanaphylactic fragments induced proliferation of human Bet v 1-specific T cell clones, indicating that they harbored all dominant T cell epitopes and therefore may be considered as a basis for the development of a safe and specific T cell immunotherapy.
Allergens↗
High-level expression and purification of the major birch pollen allergen, Bet v 1.
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.
Allergens↗
Allergenic crossreactivities. Pollens and vegetable foods.
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Allergens↗
Recombinant Bet v 1, the major birch pollen allergen, induces hypersensitivity reactions equal to those induced by natural Bet v 1 in the airways of patients allergic to tree pollen.
BACKGROUND: Atopic allergens produced by recombinant DNA methods are promising tools for diagnosis and therapy of Type I allergy. To evaluate the immunologic properties of these molecules, it is necessary to compare them with natural allergens in vitro and in vivo. OBJECTIVE: The study was carried out to determine whether the potency of recombinant Bet v 1 (rBet v 1) is comparable to that of natural Bet v 1 (nBet v 1) in inducing allergic reactions in the nose and bronchi. METHODS: Thirteen patients allergic to birch pollen with bronchial asthma and/or rhinitis were investigated. Skin prick tests and nasal and bronchial challenges were performed with rBet v 1 and nBet v 1. RESULTS: In patients allergic to birch pollen, both allergens induced comparable skin reactions. In subjects with rhinitis rBet v 1 was equally potent in inducing nasal reactions (mean PD(+60)NR +/- SD, 10.48 +/- 17.42 microg vs 7.98 +/- 8.9 microg, p > 0.05). In patients with asthma, rBet v 1 was equally potent in inducing bronchial reactions (PD20 FEV1, 0.81 +/- 1.74 microg vs 0.62 +/- 1.44 microg, p > 0.05) as nBet v 1. CONCLUSION: No significant differences were observed between natural and recombinant allergen. We conclude that allergens produced by recombinant techniques can induce typical allergic reactions in important target organs of Type I allergy: the nose and bronchi.
Adult↗
Cell surface characterization of T lymphocytes and allergen-specific T cell clones: correlation of CD26 expression with T(H1) subsets.
To evaluate the possibility of using surface molecules as markers for human T helper cell subsets, we studied the expression of surface molecules on T cell clones (TCCs) specific for the major birch pollen allergen, Bet v 1. No difference in the expression of the respective receptors for interferon-gamma (IFN-gamma), IL-4, IL-6, or IL-10 could be detected on T(H) subsets, nor did CD25 expression (IL-2R alpha-chain) differ significantly. However, high expression of CD26 antigen (dipeptidyl peptidase IV) correlated with a T(H1)/T(H0)-like phenotype, whereas T(H2)-like clones displayed a lower expression of CD26 antigen. Comparing cytokine production and CD26 expression simultaneously, we found a correlation between the IL-4/IFN-gamma ratio and the density of CD26 per cell. We could show that the amount of IL-4 secretion, and not of IFN-gamma secretion, was responsible for this correlation. To evaluate whether CD26 antigen expression is regulated by stimuli inducing a T(H1)- or T(H2)-like phenotype, peripheral blood mononuclear cells (PBMC) were cultured in the presence of IL-4, IFN-gamma, and IL-12, respectively. Incubation with IL-4 led to T cells with a T(H2)-like cytokine pattern and a significantly lower expression of CD26; IFN-gamma and IL-12 led to a T(H1) shift associated with an increased expression of CD26 on CD4+ T cells. By means of intracellular cytokine detection we analyzed expression of CD26 on CD4+ PBMC stimulated to produce IFN-gamma or IL-4 on a single cell level. All activated, cytokine-producing CD4+ T cells expressed CD26, but the increase in CD26 expression was higher in cells producing IFN-gamma. These data suggest that regulation of CD26 cell surface expression correlates with the production of T(H1)-like cytokines.
Allergens↗
Natural latex, grass pollen, and weed pollen share IgE epitopes.
BACKGROUND: Because of the frequent use of natural latex products, IgE-mediated reactions to latex proteins represent an important health threat in industrialized countries. Although several latex allergens have been characterized and IgE cross-reactivities with allergens present in plant-derived food have been described, limited information is available regarding the presence of common IgE-binding components in latex and plant pollen. METHODS: By using serum IgE from 56 individuals with latex allergy, the IgE-binding components in ammoniated latex milk and latex glove extracts were characterized by immunoblotting. The presence of cross-reactive IgE-binding components in the different latex extracts, extracts from mugwort, ragweed, timothy grass pollen, and recombinant birch pollen allergens (Bet v 1 and Bet v 2 [birch profilin]) was studied by immunoblot inhibitions and quantitative competition experiments. The involvement of carbohydrates in the constitution of cross-reactive IgE epitopes was studied by periodate treatment of extracts. RESULTS: Although sera from certain individuals with latex allergy showed IgE reactivity with protein bands of different molecular weights in Western-blotted latex milk and glove extracts, both extracts contained common IgE epitopes. Although preincubation with recombinant Bet v 1 and Bet v 2 did not significantly inhibit IgE binding to latex proteins, weed and, in particular, timothy grass pollen extract strongly inhibited IgE binding to latex allergens. The cross-reactive IgE epitopes were sensitive to periodate treatment. CONCLUSIONS: Mugwort, ragweed, and timothy grass pollen share IgE epitopes with glycoprotein latex allergens. The presence of common epitopes might in part explain clinical symptoms in patients allergic to pollen on contact with latex.
Adolescent↗
Major cat and dog allergens share IgE epitopes.
BACKGROUND: Patients allergic to cats and dogs frequently display IgE reactivity against allergens from different animals, suggesting a cross-sensitization to common allergenic determinants. Although albumins have been recognized as relevant cross-reactive allergens, little is known regarding cross-reactive epitopes of the major cat and dog allergens. OBJECTIVE: In this study, sera from patients allergic to cats and/or dogs were used to investigate the presence of common IgE epitopes among the major cat and dog allergens. METHODS: The IgE reactivity profile of 109 patients who were allergic to allergens from several species of animals was determined with nitrocellulose-blotted cat and dog allergens. Sera from patients who were strongly allergic to the major cat and dog allergens were tested for the presence of cross-reactive IgE antibodies by one-dimensional and two-dimensional immunoblot inhibition experiments and by quantitative measurements obtained with the CAP-FEIA system (Pharmacia). RESULTS: Sixty-eight of 109 patients with animal allergy showed IgE reactivity to cat allergens and dog allergens. Sera from patients with both cat and dog allergy detected allergens of similar molecular weight in nitrocellulose-blotted cat and dog hair/dander extracts. Common, as well as species-restricted, IgE epitopes of the major cat and dog allergens could be demonstrated by IgE inhibition studies. CONCLUSION: Shared IgE epitopes of the major cat and dog allergens may provide an explanation for the clinical observation that allergies to cats and dogs are frequently associated.
Albumins↗
Modulation of the allergic immune response in BALB/c mice by subcutaneous injection of high doses of the dominant T cell epitope from the major birch pollen allergen Bet v 1.
Several in vitro and in vivo studies indicate that application of high doses of dominant T cell epitopes can induce a state of antigen-specific non-responsiveness (anergy). In the present study, we developed a murine model of an allergic immune response to Bet v 1, the major birch pollen allergen. Mice were sensitized by injection of rBet v 1 and the allergic state was proven by the presence of allergen-specific IgE and positive immediate-type skin tests to Bet v 1. In epitope mapping experiments, an immunodominant T cell epitope of Bet v 1 in BALB/c mice was identified by the use of overlapping peptides. This peptide (BV 139) was subsequently employed for treatment. Two tolerization protocols were used: in one approach, the peptide was administered to naive mice before immunization (group BV139-S), in the second, already sensitized mice were treated (S-BV139). The results demonstrated that administering high doses of the dominant T cell epitope of Bet v 1 profoundly diminished T cell proliferation to the peptide in the BV139-S group, and to the peptide as well as to the whole protein in the S-BV139 group. Skin test reactivity to Bet v 1 was reduced in the BV139-S group. However, no differences in terms of specific antibody production between treated and untreated mice could be observed. This study provides evidence that administration of dominant T cell epitopes can down-regulate the allergen-specific T cell response. Proceeding on the assumption that the T lymphocyte response to allergens is crucial for the induction and maintenance of the allergic disease, a modulation of the immune response to allergens by treatment with T cell epitope peptides could represent a promising concept for immunotherapy in the future.
Adjuvants, Immunologic↗
Bet v 1, the major birch pollen allergen, conjugated to crystalline bacterial cell surface proteins, expands allergen-specific T cells of the Th1/Th0 phenotype in vitro by induction of IL-12.
Modulation of allergic immune responses by using adequate adjuvants is a promising concept for future immunotherapy of type I hypersensitivity. In the present study, recombinant Bet v 1 (rBet v 1, the major birch pollen allergen) was conjugated to cross-linked crystalline surface layer proteins (SL) derived from Gram-positive eubacteria. T cell lines (TCL) and clones (TCC) were established from peripheral blood of birch pollen-allergic patients. TCL and TCC were induced either using rBet v 1 alone or rBet v 1/SL conjugates (rBet v 1/SL) as initial antigen stimulus. Cytokine production after re-stimulation with rBet v 1 was investigated. TCL initiated with rBet v 1/SL showed significantly increased IFN-gamma production as compared to rBet v 1 -selected TCL. TCC were established from TCL of five patients. As expected, the majority of CD4+ TCC induced by rBet v 1 (55%) displayed a Th2-like pattern of cytokine production. However, only 21% of Bet v 1-specific TCC isolated from TCL established with the Bet v 1/SL revealed this phenotype. The majority of SL-specific TCC (80%) belonged to the Th1 phenotype. In cultures of peripheral blood mononuclear cells, both, SL and Bet v 1/SL (but not rBet v 1) stimulated the production of high levels of IL-12, a pivotal mediator of Th1 responses. Moreover, stimulation of rBet v 1-induced TCC with rBet v 1/SL led to an increased IFN-gamma production. This effect could be reversed by neutralizing anti-IL-12 mAb. Together these results indicate an adjuvant effect of SL mediated by IL-12. Our results indicate that bacterial components, such as SL, displaying adjuvant effects may be suitable for immunotherapeutical vaccines for type I allergy.
Adjuvants, Immunologic↗
Immunological changes during specific immunotherapy of grass pollen allergy: reduced lymphoproliferative responses to allergen and shift from TH2 to TH1 in T-cell clones specific for Phl p 1, a major grass pollen allergen.
BACKGROUND AND OBJECTIVE: The mechanisms operative in specific immunotherapy (SIT) of Type I allergy are not completely understood. In the present study we evaluated immunological changes during SIT in pollinosis. METHOD: Eight patients suffering from pollinosis (monosensitized to grass pollen) were treated with conventional SIT. All subjects had IgE specific for Phl p 1, a major allergen of timothy grass. In vitro changes in the immunological reactivity to grass pollen extract and to recombinant Phl p 1 were evaluated. Subjects were examined at three occasions: before, after 3 months and after 1 year of SIT. RESULTS: Serological analysis revealed a marked increase of grass pollen- and Phl p 1-specific IgG, titres of specific IgE did not change significantly. Lymphoproliferative responses to grass pollen extract and rPhl p 1 were reduced already after 3 months of treatment. Accordingly, the cloning efficiency for Phl p 1-specific T-cell clones (TCC) dropped markedly in all patients. The majority of allergen-specific TCC raised before SIT revealed a TH2-like pattern of cytokine production, TCC established after SIT revealed TH1 characteristics. This shift was due to a decrease in IL-4 rather than an increase in IFN-production by T cells. Investigations of the epitopes recognized by T cells before and after SIT did not reveal the outgrowth of new ('protecting') specificities. We could not observe induction of allergen-specific CD8+ lymphocytes (supressor cells). CONCLUSION: Our data indicate that -- on the level of TH lymphocytes -- SIT induces tolerance to the allergen and a modulation of the cytokine pattern produced in response to allergen stimulation.
Adult↗
Food allergy to pumpkinseed--characterization of allergens.
In recent years, pumpkinseed has become increasingly popular as a foodstuff. Here we report the occurrence of allergic reactions (itching and swelling of oral mucosa, and asthma) to this member of the Cucurbitaceae family. We investigated three patients suffering from symptoms after ingestion of roasted pumpkinseed. All the patients fished for sport and used pressed pumpkinseed flour as bait. Sera were tested by the immunoblot technique for IgE reactivity with proteins of pumpkinseed extract. The immunoblot revealed pumpkinseed allergens of 13, 14, 36, 48, 77, and 87 kDa. Inhibition experiments with recombinant birch profilin were performed: IgE binding to the 14-kDa allergen was completely blocked by preincubation of the sera with recombinant birch profilin. In conclusion, type I allergy to pumpkinseed is rare, and the patients' histories suggest inhalation of pumpkinseed flour during fishing to be the relevant route of sensitization, leading to food allergy to pumpkinseed.
Adult↗
Heterogeneity in the polyclonal T cell response to birch pollen allergens.
BACKGROUND: Immunodominant epitopes of Bet v 1a had been identified before, using recombinant (r) Bet v 1a-reactive T cell clones generated from peripheral blood mononuclear cells of patients allergic to birch pollen. This study aimed at evaluating the T cell-stimulating capacity of immunodominant Bet v 1a-derived peptides in a polyclonal system corresponding more closely to the situation in patients. METHODS: Short-term T cell lines (TCL) were established in presence of a protein extract of birch pollen (BP extract). TCL proliferation induced by the BP extract, by natural Bet v 1, rBet v 1a, rBet v 2 or 5 selected immunodominant Bet v 1a-derived peptides was determined. RESULTS: Consistent with the knowledge that Bet v 1 is the major IgE-binding allergen of birch pollen, we found comparable T cell reactivity to natural Bet v 1 and the BP extract within the majority of the TCL. Accordingly, the response to rBet v 2 was low compared with the reactivity to the BP extract. The response of the TCL to rBet v 1a proved to be highly heterogeneous. Furthermore, the TCL response to the 5 immunodominant Bet v 1a-derived peptides showed considerable diversity. The proliferative responses of most TCL (with one exception) following stimulation by these peptides were low, in relation to the expansion induced by the BP extract. CONCLUSION: These findings argue against the use of selected peptides derived from Bet v 1a in specific immunotherapy of patients with birch allergy.
Allergens↗
Persistent pruritus after hydroxyethyl starch infusion therapy: a result of long-term storage in cutaneous nerves.
A high incidence of severe pruritus has been observed after the administration of hydroxyethyl starch (HES) on account of plasma volume substitution and improvement of the microcirculation. The aim of this study was to elucidate the possible pathomechanisms of HES-induced itching. Skin biopsies were taken from 93 patients, half of them presenting with pruritus, who received HES of various preparations and cumulative dosages. The samples were subjected to immunoelectron microscopical investigation using an antibody highly specific for HES. After infusion therapy with HES, formation of intracytoplasmic storage vacuoles in the skin could be demonstrated in all patients. A dose-dependent uptake of HES was first detectable in macrophages and, thereafter, in endothelial and epithelial cells. Consecutive control biopsies taken from single patients revealed a subsequent reduction of the vacuoles, in size and number, within 3 years, thus indicating a regular cutaneous metabolism of HES. Patients suffering from pruritus consistently showed additional deposition of HES in small peripheral nerves. HES-reactive vacuoles could be demonstrated in the Schwann cells of unmyelinated, as well as small myelinated, nerve fibres, and in endoneural and perineural cells. Neural devacuolization paralleled the clinical improvement in the symptoms. In conclusion, HES deposits in cutaneous nerves, as a consequence of a higher cumulative dosage, may account for the itching seen after HES infusion.
Adolescent↗