Biomedical subjects

R Valenta

Publications and source records attributed to R Valenta.

At least 163 records · Page 9Linked to original sources

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

Immunogold electron microscopic localization of timothy grass (Phleum pratense) pollen major allergens Phl p I and Phl p V after anhydrous fixation in acrolein vapor.

We used the vapor phase of acrolein as an anhydrous fixative for timothy grass pollen in an immunogold double-labeling localization study of two different major allergens, Phl p I and Phl p V. More than 48 hr of fixation were needed for the subcellular pollen structures to be satisfactorily stabilized. The immunoreactivity of acrolein-fixed pollen allergens was not destroyed even after prolonged acrolein fixation. By immunoblotting, the two allergens differ in their immunological and structural characteristics. Electron microscopic localization traced the allergens at least partially to different subcellular pollen compartments.

Acrolein↗

PubMed1994

Molecular and functional characterization of allergens: basic and practical aspects.

Well-defined allergen preparations are, in the first line, a prerequisite for exact diagnosis, but will be supposedly useful tools in immunotherapy of Type I (IgE-mediated) allergic diseases. The allergens have to be available in standardized and highly purified form in sufficient quantities. By applying recombinant DNA techniques this goal can be achieved with respect to both, characterization and reproducibility of allergen preparations. As an example, purified recombinant non-fusion Betv1 revealed identical immunological properties with respect to interaction with both, anti-Betv1 antibodies and Betv1-specific T cell clones when compared with natural Betv1 purified from birch pollen. Moreover, cloning of allergens yielded a number of deduced primary structures of allergens, which allows computer-aided comparisons with already known amino acid sequences. Significant sequence similarities with well-described proteins may point at a biological and biochemical function of the cloned allergen, which might be of interest for considerations why a certain protein within an extract represents an allergen. Furthermore, the interaction of small peptides synthesized according to amino acid sequences of cloned allergens with allergen-specific T and B cells can be investigated. Respective results will yield information about the regulation of IgE synthesis and, thus, might point at new concepts of immunotherapy.

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

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

Identification of multiple T cell epitopes on Bet v I, the major birch pollen allergen, using specific T cell clones and overlapping peptides.

Eleven T cell clones (TCC) with specificity for Bet v I were established from the peripheral blood of six birch pollen allergic donors. Bet v I is the major allergen of birch (Betula verrucosa) pollen and shows high homology to the major allergens of pollens of other trees within the order fagales (hazel, alder, hornbeam, oak, etc.), which represent important inhalant allergens in the northern hemisphere. The TCC were shown to react with purified natural, as well as with purified recombinant Bet v I. All clones showed the helper cell phenotype (CD3+CD4+) and expressed the TCR-alpha/beta. The cytokine production pattern in response to stimulation with allergen resulted in enhanced production of IL-4 in 9 of 11 clones. The clones were used for T cell epitope mapping on the Bet v I molecule. For this purpose, peptides with a length of 12 amino acids each and overlapping for 10 residues were synthesized following the amino acid sequence of Bet v I. These 75 peptides were used to stimulate Bet v I-specific T cell clones. Our experiments revealed 7 distinct T cell epitopes on the Bet v I molecule. The epitopes were scattered over the whole molecule, 2 sequences were in agreement with an algorithm previously described for the prediction of T cell epitopes. In 3 cases, we could identify distinct TCC specificities within single individuals. Furthermore, for each donor, none of the peptides representing epitopes for TCC inhibited the binding of IgE antibodies to Bet v I. These results suggest that T cells and IgE antibodies from the same individual recognize different structures on the Bet v I allergen.

Allergens↗

PubMed1993

Induction of specific histamine release from basophils with purified natural and recombinant birch pollen allergens.

As much as 15% of the population in industrialized countries suffers from type I allergic symptoms (rhinitis, conjunctivitis, and bronchial asthma). One approach toward this disease involves the production of recombinant allergens in Escherichia coli and their purification for diagnostic and therapeutic purposes. In this study we compared the IgE-binding capacity of natural and recombinant birth allergens with their functional ability to release histamine from allergic patients' basophils via cross-linking of high-affinity Fc epsilon-receptors. The recombinant as well as pollen-derived Bet v I and birch profilin (Bet v II) were purified and tested in parallel on IgE immunoblots and in in vitro histamine release tests (n = 21). We observed an excellent correlation between allergen-induced histamine release and birch pollen RAST (r = 0.881, p < 0.002). Nonspecific histamine release as a result of a cell toxic effect of the allergen preparations was never observed from basophils of donors without birch pollen allergy. The specificity of the presented effector model is also documented by the specific desensitization of patients' basophils with the recombinant allergens. These data may provide a basis for the use of purified recombinant allergens in sensitive and specific in vitro allergy tests monitoring the effector situation in allergic patients, which therefore may represent a possible alternative for in vivo diagnostic methods.

Allergens↗

PubMed1993

The profilin multigene family of maize: differential expression of three isoforms.

Profilin is a small (12-15 kDa) actin- and phospholipid-binding protein previously known only from studies on animals and lower eukaryotes but recently identified as a birch pollen allergen. Here we have identified and characterized three members of the profilin multigene family from the plant Zea mays. Two cDNAs isolated from a maize pollen library (ZmPRO 1 and ZmPRO 3) each have a single, large open reading frame encoding a putative polypeptide 131 amino acids long with a predicted molecular weight of approximately 14 kDa. A third maize pollen cDNA (ZmPRO 2) has two in-frame translation initiation codons. Use of the first ATG would result in a polypeptide 137 amino acids long with a molecular weight of 14.8 kDa. The three maize profilins are highly homologous to each other (> 90% nucleotide and amino acid sequence identity) as well as other plant profilins but show far less similarity (30-40% amino acid sequence identity) to animal and lower eukaryote profilins. Multiple sequence alignments indicate that only nine residues are shared by all eukaryotic profilins examined. However, limited comparisons reveal domains in the NH2 and COOH termini that have a high degree of similarity suggesting functional conservation. The maize gene family size is estimated to contain three to six members based on Southern blot experiments with gene-specific and coding region probes. Northern blot analysis demonstrates that the three maize profilin cDNAs characterized here are utilized in a tissue-specific manner and are anther or pollen specific.

Actins↗

PubMed1993

Allergens from birch pollen and pollen of the European chestnut share common epitopes.

Type I allergy to pollen of the European chestnut (Castanea sativa) represents a major cause of pollinosis in (sub) Mediterranean areas. Using sera from 14 patients with established allergy to pollen of the European chestnut, 13/14 sera (92%) showed IgE-binding to a 22 kD protein, 2/14 (14%) displayed additional binding to a 14 kD protein and 1/14 (7%) bound only to the 14 kD protein of European chestnut pollen extract. Two monoclonal mouse antibodies, BIP 1 and BIP 4, directed against different epitopes of Bet v I (the major birch pollen allergen), and a rabbit antibody to recombinant birch profilin (rBet v II) were used to characterize the proteins of the European chestnut pollen. The recombinant birch pollen allergens, rBet v I and rBet v II (profilin) were employed to show common allergenic structures on proteins from both birch and European chestnut pollen by IgE-inhibition experiments. Despite the fact that the 22 kD protein displayed a higher molecular weight in comparison to the 17 kD major birch pollen allergen, Bet v I, we could demonstrate reactivity of both monoclonal antibodies, BIP 1 and BIP 4, with this protein. A complete inhibiton of IgE-binding to this 22 kD protein was shown by pre-incubating sera with purified recombinant Bet v I. In addition, the 14 kD protein could be identified by IgE-inhibition studies with recombinant Bet v II and by using a rabbit anti-profilin antibody as the profilin from pollen of the European chestnut.

Allergens↗

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

[Effector cells in allergy: biological principles and new pharmacologic concepts].

The clinical symptoms of allergy are caused by cellular (IgE-triggered) responses to an allergen. Effector cells of allergy include eosinophil and basophil granulocytes, as well as tissue mast cells. Growth and accumulation, as well as IgE-dependent and independent functions of these cells are regulated by distinct proteohormones and peptides. The hemopoietic cytokines IL-3 (interleukin-3), IL-5 and GM-CSF (granulocyte-macrophage colony-stimulating factor) are involved in the regulation of basophils (and eosinophils), whereas the ligand for c-kit, SCF (stem cell factor) is a mast cell-specific agonist. Basophils and mast cells express high-affinity IgE-binding sites. Allergen binding to IgE on mast cells and basophils, and consecutive cross-linking of IgE receptors is followed by production and/or secretion of inflammatory mediator substances. Specific activation and deactivation of mast cells/basophils in vitro has been demonstrated by use of recombinant cytokines and allergens, and specific haptens or by use of novel drugs, and should lead to epitope-specific diagnosis and better management of allergic diseases in the future.

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↗

PubMed1993

Tumor necrosis factor alpha and interleukin-1 beta mRNA expression in HMC-1 cells: differential regulation of gene product expression by recombinant interleukin-4.

Cytokine-activation pathways in mast cells are supposed to play a significant role in host defense mechanisms and allergic reactions. Interleukin-4 (IL-4) is a well-characterized regulator of growth and function of mast cells. The human mast cell line HMC-1 was established from a patient suffering from mast cell leukemia and was shown to expose IL-4 binding sites. In the present study, the effects of recombinant human (rh) IL-4 and other rh cytokines (IL-2, IL-3, IL-6, IL-8) on expression of cytokine mRNA in HMC-1 cells were examined by Northern blot analysis using oligonucleotide probes. Tumor necrosis factor alpha (TNF-alpha) and IL-1 beta transcripts were found to be expressed constitutively in HMC-1 cells, whereas transcripts for IL-3, IL-4, IL-5, IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF) could not be detected. Of all cytokines tested, rhIL-4 was found to down-regulate IL-1 beta mRNA expression and formation of immunoreactive IL-1 beta protein in HMC-1 cells. The effect of IL-4 on IL-1 beta gene product expression was time- and dose-dependent (maximum effects obtained with 100 U/mL of rhIL-4). No effect of IL-4 on expression of TNF-alpha mRNA in HMC-1 cells was observed. These results raise the possibility that human mast cells are a source of both TNF-alpha and IL-1 beta. Furthermore, our study provides evidence that IL-4 regulates IL-1 beta gene product expression in HMC-1 cells. The HMC-1 cell line should be a useful tool for studying cytokine activation pathways in human mast cells.

Base Sequence↗

PubMed1992

Profilins constitute a novel family of functional plant pan-allergens.

Type I allergy is a major health problem in industrialized countries where up to 15% of the population suffer from allergic symptoms (rhinitis, conjunctivitis, and asthma). Previously, we identified a cDNA clone that encoded a birch pollen allergen as profilin. Profilins constitute a ubiquitous family of proteins that control actin polymerization in eukaryotic cells; in particular, profilin participates in the acrosomal reaction of animal sperm cells. Although profilins had been unknown in plants so far, our finding led to the assumption that profilins might have similar functions in pollens during plant fertilization and therefore represent allergenic components in almost all pollens. We show that profilins are prominent allergens that can be isolated from tree pollens (Betula verrucosa, birch), from pollens of grasses (Phleum pratense, timothy grass), and weeds (Artemisia vulgaris, mugwort). About 20% of all pollen allergic patients tested (n = 65) displayed immunoglobulin E (IgE) reactivity to recombinant birch profilin that was expressed in pKK223-3. An IgE inhibition experiment performed with recombinant birch profilin and purified natural profilins from timothy grass and mugwort indicates common IgE epitopes. Moreover, all pollen profilins purified from these far distantly related plant species, and likewise the purified recombinant birch profilin, are able to elicit dose-dependent histamine release via high affinity Fc epsilon receptor of blood basophils from profilin allergic patients. The presence of profilin and possibly related proteins as crossreacting allergenic components in various plants therefore provides an explanation as to why certain allergic patients display type I allergic reactions with pollens and even food from distantly related plants. A functional pan-allergen, like profilin, available as purified recombinant protein, may be a useful diagnostic and probably therapeutic reagent.

Allergens↗

PubMed1992

Complementary DNA cloning and expression in Escherichia coli of Aln g I, the major allergen in pollen of alder (Alnus glutinosa).

Previous data showed that the major pollen allergens from trees of the order Fagales, in particular alder, birch, hazel, and hornbeam, are highly interrelated. As only the complete amino acid sequence of Bet v I, the major allergen from birch, has been known, it was of interest to obtain the primary structure of other major allergens of this group, to attribute IgE-binding properties to certain features of the amino acid sequences of those allergens. cDNA was synthesized from alder pollen mRNA, sequence-specifically amplified by polymerase chain reaction and cloned into plasmid bluescript. Comparison of the deduced amino acid sequences of Aln g I and Bet v I revealed a 86.8% homology. The Aln g I encoding cDNA was subcloned into pKK223-3 and expressed in Escherichia coli as a full-length nonfusion protein. The recombinant Aln g I bound IgE from tree pollen-allergic patients and was shown to share IgE-epitopes with Bet v I by inhibition studies with recombinant Bet v I. Computer-aided calculations predicted epitopes in both Aln g I and Bet v I at the same position; the Bet v I molecule was predicted to possess two additional epitopes near the N-terminus of the molecule.

Allergens↗