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S Vrtala

Publications and source records attributed to S Vrtala.

At least 55 records · Page 3Linked to original sources
PubMed1996

Immunologic characterization of purified recombinant timothy grass pollen (Phleum pratense) allergens (Phl p 1, Phl p2, Phl p 5).

BACKGROUND: Grass pollen allergens belong to the potent elicitors of type I allergy. Approximately 40% of allergic individuals display IgE reactivity with grass pollen allergens. In previous studies we have reported the complementary DNA cloning and expression in Escherichia coli of three of the most relevant timothy grass pollen allergens: Phl p 1, Phl p 2, and Phl p 5. OBJECTIVE: To achieve high level expression of immunologically active timothy grass pollen allergens in E. coli, the cDNAs were inserted into expression plasmids. METHODS: The three recombinant grass pollen allergens were expressed at high levels in E. coli as recombinant nonfusion proteins, purified by conventional protein chemical methods and tested for their IgE-binding capacity by immunoblot and ELISA, as well as in histamine release assays. RESULTS: Milligram amounts of pure recombinant allergens were obtained from cultured E. coli. IgE binding to purified recombinant Phl p 1, Phl p 2, and Phl p 5 could be demonstrated by immunoblot and ELISA. With ELISAs the percentage of grass pollen-specific IgE directed against the individual recombinant allergens could be estimated. In addition, the purified recombinant timothy grass pollen allergens induced dose-dependent and specific histamine release from patients' blood basophils. CONCLUSION: Purified recombinant timothy grass pollen allergens represent useful tools for diagnosis and therapy of grass pollen allergy.

Allergens↗
PubMed1994

Isolation of an immunodominant IgE hapten from an epitope expression cDNA library. Dissection of the allergic effector reaction.

An epitope expression cDNA library was constructed from the randomly fragmented cDNA coding for Phl p I, the major grass pollen allergen. Using IgE from allergic patients, epitope clones were isolated and immunodominant fragments were selected. Among three epitope clones coding for a similar region of Phl p I, one clone expressed a 15-amino-acid epitope which was target for IgE antibodies from approximately 30% of grass pollen allergic patients. According to the prevalence of grass pollen allergy, 22% of all allergic patients are expected to display IgE reactivity with this epitope. Although the purified recombinant epitope specifically bound IgE, it did not release histamine from basophiles of most grass pollen allergic patients and thus represents an IgE hapten. Immunodominant IgE haptens may be useful as therapeutic agents to saturate mast cell-bound IgE prior to allergen exposure and may represent candidates for a safe immunotherapy of allergic diseases by reducing anaphylactic side effects.

Allergens↗
PubMed1994

cDNA cloning and expression of timothy grass (Phleum pratense) pollen profilin in Escherichia coli: comparison with birch pollen profilin.

Profilin, an actin-binding protein, was previously described as a ubiquitous allergen which is responsible for cross-reactivities in about 20% of pollen and food allergic patients. A complete cDNA clone coding for timothy grass (Phelum pratense) pollen profilin was isolated using allergic patients IgE. The deduced amino acid sequence of timothy grass profilin shares a sequence identity of 79% with birch profilin and other plant profilins and a lower average sequence identity of 35% with other eukaryotic profilins. The high degree of homology among different plant profilins at the DNA and protein level explains the extensive cross-reactivities observed in profilin allergic patients. Recombinant timothy grass pollen profilin was expressed in Escherichia coli as a beta-galactosidase fusion protein and shown to bind IgE from profilin allergic patients similar to recombinant birch profilin. Slight differences regarding the IgE-binding capacity of birch and timothy grass profilin indicate that not all IgE-epitopes of the two profilins are conserved. It is speculated that profilin allergic patients were initially sensitized against a certain profilin and then cross-react with the homologous proteins.

Allergens↗
PubMed1994

IgE-binding capacity of recombinant timothy grass (Phleum pratense) pollen allergens.

A panel of 60 cDNA clones coding for IgE-binding proteins from timothy grass pollen was immunocharacterized with sera from 30 patients allergic to grass pollen and antibodies raised against natural grass pollen allergens. In the cases of five representative patients in whom the IgE reactivity pattern with the recombinant allergens had been determined, IgE immunoadsorption experiments were performed. Recombinant Phl p I, Phl p V, and Phl p II and recombinant timothy grass profilin were used for immunoadsorption of the sera, and the percentage of remaining grass pollen-specific IgE was estimated. Although most of the patients showed IgE reactivity to a number of different natural and recombinant timothy grass pollen allergens, up to 66% of IgE directed against blotted total natural grass pollen allergens could be immunoadsorbed from the sera with recombinant Phl p V and Phl p I. The data point to the usefulness of recombinant allergens not only to determine IgE specificities of allergic patients but also to estimate the percentage of specific IgE that individuals produce against certain allergens. The fact that only a limited number of recombinant timothy grass pollen allergens account for a high percentage of grass pollen-specific IgE points to the possible usefulness of recombinant allergens not only for in vitro diagnosis but probably also for specific immunotherapy.

Allergens↗
PubMed1994

Complementary DNA cloning of the major allergen Phl p I from timothy grass (Phleum pratense); recombinant Phl p I inhibits IgE binding to group I allergens from eight different grass species.

BACKGROUND: Grass pollens, such as pollen from timothy grass (Phleum pratense), represent a major cause of type I allergy. OBJECTIVE: In this report we attempted to determine how cross-reactive allergenic components of grass pollens from different species can be represented by a minimum number of recombinant allergens. METHODS: We isolated and sequenced a timothy grass pollen cDNA coding for the major allergen Phl p I. A recombinant Phl p I-beta-galactosidase fusion protein, which bound to IgE in 87% of patients with grass pollen allergy, was produced in Escherichia coli. Using recombinant Phl p V and Phl p I, we defined representative patients' sera that bound to group I but not to group V allergens, as well as sera with reactivity against group I and group V allergens. IgE immunoblot inhibition studies were done with nitrocellulose-blotted pollen extracts from eight grass species with different geographic distribution. RESULTS: Preadsorption of patients' sera with recombinant nonfusion Phl p I strongly reduced IgE binding to group I allergens from the eight grasses, showing extensive cross-reactivity between species. CONCLUSION: A single recombinant group I allergen contains many of the IgE epitopes of group I isoallergens from a number of different grass species.

Allergens↗
PubMed1994

B-cell epitopes of allergens determined by recombinant techniques; use for diagnosis and therapy of type I allergy.

In the present manuscript, the immunological and functional in vitro properties of recombinant plant allergens are summarized. Recombinant tree pollen allergens (major birch pollen allergen-BetvI, birch profilin-BetvII) and recombinant timothy grass pollen allergens (PhlpI, PhlpV, and PhlpII) were compared with the natural counterparts regarding IgE-binding properties and capacity to release histamine from patients' basophils. In addition, experimental in vivo models of Type I allergy, based on recombinant allergens, are discussed. The major conclusion is that recombinant allergens can be seriously considered as candidates for diagnosis of Type I allergy allowing to establish specific allergograms for the individual patients. The in vivo data obtained in mouse and primate systems indicate that recombinant allergens can be used to set up close-to-man models of Type I allergy. Such in vivo models are useful to test the effects of already established therapeutic approaches and also allow to develop therapeutical concepts which are based on the use of recombinant allergens. Examples of specific therapeutical concepts are presented.

Allergens↗
PubMed1993

Molecular characterization of Phl p II, a major timothy grass (Phleum pratense) pollen allergen.

Grass pollen allergens belong to the most important and widespread elicitors of pollen allergy. Using serum IgE from a grass pollen allergic patient, a complete cDNA encoding a group II allergen was isolated from a timothy grass (Phleum pratense) pollen expression library. The deduced amino acid sequence of the Phl p II allergen shows an average sequence identity of 61% with the protein sequences determined for group II/III allergens from rye grass (Lolium perenne) and a sequence identity of 43% with the C-terminal portion of group I grass pollen allergens from different species. A hydrophobic leader peptide similar to leader peptides found in other major grass pollen allergens heads the deduced amino acid sequence, indicating that group II/III grass pollen allergens belong to a family of secreted proteins. Serum IgE specific for Phl p II, detected the protein exclusively in pollen and not in other plant tissues. The recombinant Phl p II was expressed in Escherichia coli and showed similar IgE-binding capacity as the natural allergen.

Allergens↗
PubMed1993

cDNA cloning of a major allergen from timothy grass (Phleum pratense) pollen; characterization of the recombinant Phl pV allergen.

We isolated a cDNA encoding a major grass pollen allergen from a timothy grass (Phleum pratense) pollen expression cDNA library using allergic patients' IgE. The complete cDNA encoded an allergen that binds IgE from about 80% of grass pollen-allergic patients. Significant sequence homology was found to other major grass pollen allergens from Kentucky bluegrass (Poa pratense) as well as from rye grass (Lolium perenne) which originally were believed to form different identities. Using different monoclonal and polyclonal antibodies raised against group V allergens we identified the recombinant protein as a group V allergen from timothy grass, Phl p V. In IgE-binding studies it is demonstrated that the rPhl p V allergen can be used to block binding of patients' IgE to natural group V isoallergens on two-dimensional immunoblots. IgE inhibition experiments show that up to 60% of grass pollenspecific IgE can be preadsorbed with the rPhl p V allergen from patients sera. The purified rPhl p V induced specific histamine release of blood basophils from grass pollen-allergic patients. This emphasizes the usefulness of the rPhl p V for diagnostic and therapeutic purposes and corroborates the view that specific diagnosis and therapy of type l allergy could be performed with a limited panel of relevant recombinant allergens.

Amino Acid Sequence↗
PubMed1993

Identification of profilin as an actin-binding protein in higher plants.

Profilin is a low molecular weight protein involved in the organization of the mammalian and protozoan cytoskeleton as well as in signal transduction. In this study, profilin is identified as an actin-binding protein in higher plants which is present in monocot and dicot angiosperms. Birch pollen profilin and actin can be copurified as a complex, and purified recombinant birch profilin can be used as an affinity matrix to obtain birch pollen actin. The binding of 125I-labeled recombinant birch pollen profilin to plant and animal actins can be blocked by profilin-specific antibodies that react with different epitopes of birch profilin. One of the blocking antibodies was raised against the 25 COOH-terminal amino acids indicating the importance of this region in the profilactin complex formation.

Actins↗
PubMed1993

Properties of tree and grass pollen allergens: reinvestigation of the linkage between solubility and allergenicity.

In this study we reinvestigated the kinetics of allergen release from birch pollen (Betula verrucosa) and timothy grass pollen (Phleum pratense) using different protein extraction procedures, immunoblotting with specific antibodies and immune electron microscopy. Pollen allergens such as the major birch pollen allergen, Bet v I, the major timothy grass pollen allergens, Phl p I and Phl p V, group-II/III allergens from timothy grass and profilins were released rapidly and in large amounts from hydrated pollen. Within a few minutes pollen allergens could be detected in aqueous supernatants prepared from birch and grass pollen with serum IgE or specific antibodies. In parallel the allergen content in the pollen pellet fractions decreased. A nonallergenic protein such as heat shock protein 70 can be extracted in sufficient amounts only with harsh extraction procedures. Immune electron microscopy of dry and rehydrated birch pollens showed that after short hydration, the major birch pollen allergen, Bet v I, migrated into the exine and to the surface of intact pollen grains, whereas profilin, against which a lower percentage of patients is sensitized, was retained in the pollen grain. Comparing the amino acid composition and hydrophilicity of the tested allergens with a nonallergenic protein such as heat shock protein 70, no significant difference was noted. In agreement with earlier observations we conclude that the allergenic properties of proteins are rather linked to the amount and speed of solubility from airborne particles than to intrinsic properties.

Allergens↗
PubMed1993

Monitoring of two allergens, Bet v I and profilin, in dry and rehydrated birch pollen by immunogold electron microscopy and immunoblotting.

Dry and rehydrated birch pollen grains were anhydrously fixed and double immunogold-labeled for the presence of two allergens, Bet v I major allergen (17 KD) and profilin (14 KD). In dry pollen grains, both allergens are found exclusively inside the cytoplasm. In pollen grains rehydrated for 1 min, the cytoplasm is partially devoid of the two allergens, whereas the pollen wall and the germination aperture are specifically labeled. Pollen grains rehydrated for 5 min are largely free of the two allergens. In immunoblot experiments, both allergens could be detected in the aqueous supernatants of rehydrated pollen samples within 5 min. The results obtained by both methods show the high solubility of both proteins. This makes them readily available to the immune system and characterizes them as potent allergens. Moreover, the solubilization of profilin might indicate a dissociation of the profilin-actin complex at the very first stage of pollen germination, which could favor formation of the cytoskeleton and pollen tube growth.

Allergens↗
PubMed1993

[New concepts in therapy of type I allergic diseases].

The molecular characterization of allergens with recombinant DNA techniques allowed cDNA sequences to be obtained and, hence, information regarding the primary structure of allergens. It is now possible to express well-defined recombinant allergens in heterologous expression systems and to obtain large amounts of highly pure recombinant allergens for the improvement of current diagnosis and therapy of type I allergic diseases. Due to extensive cross-reactivities and structural similarities of the relevant allergens it is possible to define a limited number of allergens, which is a prerequisite for allergen-specific therapeutic concepts. Specific concepts of active immunotherapy and strategies of passive therapeutic interference based on recombinant techniques are discussed.

Allergens↗
PubMed1992

Diagnosis of grass pollen allergy with recombinant timothy grass (Phleum pratense) pollen allergens.

In order to establish a test system for grass pollen allergy based on the use of recombinant allergens we chose timothy grass (Phleum pratense), a widely spread grass, as a model. From a lambda gt11 cDNA expression library that we had constructed from pollen RNA of timothy grass (P. pratense), we had obtained with serum IgE from a grass pollen-allergic individual 60 IgE-binding clones. By differential testing with sera from different grass pollen-allergic patients, we selected three distinct clones encoding Phl p I (group I), Phl p V (group V) and profilin from timothy grass, which when used together allowed the diagnosis of grass pollen allergy in 97 out of 98 tested grass pollen-allergic patients employing a simple plaque lift technique. This recombinant test based on plaque lifts containing allergen-beta-galactosidase fusion proteins was compared with IgE immunoblots using crude pollen protein extracts from timothy grass. Both methods were in good agreement with RAST scores and clinical data, and proofed to be useful for the diagnosis of grass pollen allergy. Our results further indicate that a limited panel of only two recombinant grass pollen allergens, Phl p I and Phl p V, together with the plant panallergen profilin could be sufficient for the diagnosis and possibly immunotherapy of grass pollen allergy.

Adolescent↗
PubMed1991

Recombinant allergens for immunoblot diagnosis of tree-pollen allergy.

Diagnosis of type I allergy essentially depends on the availability of defined allergens, which can be provided by recombinant deoxyribonucleic acid (DNA) technology. We have previously isolated the c(complementary)DNAs encoding the major birch-pollen allergen, Bet v I, and another allergen with a molecular weight of 14 kd that was identified as birch profilin and designated Bet v II. These cDNAs were isolated from a lambda gt11 expression library by screening with the serum IgE from allergic patients. To obtain expression in Escherichia coli of recombinant allergens without additional fused polypeptides, both cDNAs were inserted into the plasmid pKK223-3. E. coli cells expressing Bet v I and birch profilin (Bet v II) were used for the preparation of recombinant proteins. These proteins were tested for their IgE-binding properties on immunoblots with sera from 100 different birch pollen-allergic patients. All patients' sera, which reacted with the natural allergens, Bet v I and Bet v II, demonstrated an identical IgE-binding pattern to recombinant birch-pollen allergens. Recombinant allergens may therefore be useful for the setup of diagnostic tests that allow the discrimination of different IgE-binding patterns as well as for patient-tailored immunotherapy.

Allergens↗
PubMedDate not supplied

The importance of recombinant allergens for diagnosis and therapy of IgE-mediated allergies.

In the past 10 years, a considerable number of cDNAs coding for allergens have been isolated and expressed. Intensive investigations showed that recombinant allergens and their respective natural counterparts possess comparable properties with respect to structure, function and interaction with the immune system. Recent studies documented that in vitro as well as in vivo diagnosis of IgE-mediated allergic diseases can be successfully improved by the application of recombinant allergens. In addition, new strategies for a safer specific immunotherapy (SIT) have been developed based on the knowledge of the primary structures of allergens. Naturally occurring isoforms of allergens as well as recombinant allergens with modified amino acid sequences show very low IgE binding capacity but strong T cell-stimulatory activity and represent possible candidates. In case of Bet v 1, the major birch pollen allergen, isoforms d, g and l and a Bet v 1a mutant, produced by site-directed mutagenesis resulting in 6 amino acid exchanges, fulfilled the above mentioned criteria. In a third approach, two adjacent peptides covering the entire Bet v 1a sequence were produced in an Escherichia coli expression system. These peptides contained most of the relevant T cell epitopes, but lost their IgE binding capacity and, thus, their ability to activate mast cells and basophils of sensitized patients. Our results suggest that allergen variants (isoforms, mutants, T cell epitope-containing peptides) may be used as 'hypoallergenic agents' in SIT.

Allergens↗