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Biomedical subjects

O Scheiner

Publications and source records attributed to O Scheiner.

At least 127 records · Page 7Linked to original sources
PubMed1994

Characterization of a birch pollen allergen, Bet v III, representing a novel class of Ca2+ binding proteins: specific expression in mature pollen and dependence of patients' IgE binding on protein-bound Ca2+.

A cDNA coding for a birch pollen allergen, Bet v III, with significant sequence homology to Ca2+ binding proteins was isolated from an expression cDNA library using serum IgE from a patient who was allergic to pollen. The deduced amino acid sequence of the pollen allergen contained three typical Ca2+ binding sites. Peptides mimicking the Ca2+ binding sites of Bet v III were synthesized and shown to bind 45Ca in blot overlays. The binding of patients' IgE to the recombinant allergen depended on the native protein conformation and protein-bound Ca2+. Depletion of Ca2+ led to a reversible loss of the IgE binding thus representing a conformational IgE epitope adopted by a polypeptide upon Ca2+ binding. By RNA hybridization it was demonstrated that Bet v III is expressed preferentially in mature pollen. Bet v III therefore represents a pollen allergen which because of its unique structural features also belongs to a novel class of Ca2+ binding proteins.

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

Egg yolk alpha-livetin (chicken serum albumin) is a cross-reactive allergen in the bird-egg syndrome.

Thirty-one patients with clinical history of egg allergy, bird allergy, or bird and egg allergy were investigated with the use of the immunoblot technique to compare IgE-binding components in bird feather and egg yolk and white extracts. Patients were classified into three groups according to clinical history, skin prick test results, and RAST results. Patients in group I were sensitized to bird feathers and egg yolk, patients in group II to egg white, and patients in group III to bird feather but not to eggs. Patients with bird-egg syndrome were mainly female adults, whereas egg white allergy was mainly observed in children without any obvious sex predisposition. IgE from patients with bird-egg syndrome recognized a 70 kd protein in egg yolk (chicken serum albumin = alpha-livetin) and some major allergens in bird feather extract (70, 95, and 200 kd). Preincubation of pooled sera from patients with bird-egg syndrome with budgerigar or hen feather extract and egg yolk extract, respectively, led to complete blocking of IgE binding to allergens in egg yolk and bird feather extract. On the other hand, IgE from patients with egg white allergy did not react with allergens in egg yolk and bird feather extract, despite strong IgE binding to egg white allergens. Patients in group III displayed no reactivity to bird feather or egg allergens. Our results demonstrate common epitopes of budgerigar and hen feather and egg yolk alpha-livetin. Therefore we assume that alpha-livetin (chicken serum albumin) leads to a cross-sensitization and consequently to the "bird-egg syndrome."

Adolescent↗
PubMed1994

Molecular characterization of dog albumin as a cross-reactive allergen.

Indoor allergens comprise a group of allergenic proteins that are commonly derived from house dust mite and cat and dog dander. In addition to the two major dog allergens (molecular weights: 19 and 23 kd), dog albumin represents an important allergen for up to 35% of patients who are allergic to dogs. In IgE immunoblot inhibition studies and histamine release tests it has been demonstrated that patients who react to dog albumin exhibit IgE reactivity with purified albumins from cat, mouse, chicken, and rat. The proportion of dog-specific IgE directed against dog albumin was determined for patients allergic to dog albumin, and it ranges from 70% to 90%. By IgE immunoscreening of a lambda gt11 expression library from a dog salivary gland, we identified a number of reactive complementary DNA clones. All patients with IgE reactivity against natural dog albumin displayed IgE reactivity to the beta-galactosidase fusion protein encoded by clone 54c, which was therefore assumed to contain major IgE epitopes of dog albumin. The deduced amino acid sequence of clone 54c was compared with the Swiss-Prot library, and significant sequence homologies were found with albumins from different species (human: 82.6%, pig: 81.8%, cattle: 77.3%, sheep: 78.8%, mouse: 75.8%, and rat: 76.2%). Several other IgE-positive clones hybridized with oligonucleotides that were prepared according to this sequence. Partial complementary DNA coding for dog albumin fragments may be considered a useful tool for further characterization of major IgE epitopes of dog albumin.

Allergens↗
PubMed1994

Distribution of immunoglobulins in squamous cell carcinoma of the head and neck.

The immune response with respect to immunoglobulin production in the tumor was investigated in 23 patients with advanced squamous cell carcinomas of the head and neck. Immunohistochemical staining with monoclonal antibodies against IgG, IgM, IgA, IgD and IgE in the tumor was compared to normal hypopharyngeal mucosa. For IgG, IgA and IgM no significant differences between tumor and control tissues could be found. In contrast, a high number of IgE-positive cells was counted in most squamous cell carcinomas compared to normal mucosa. Most of these cells appeared as plasma cells. Regarding IgD the differences between tumor and control tissues, were less pronounced but also significant.

Carcinoma, Squamous Cell↗
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

Purification, characterization and N-terminal amino acid sequence of a new major allergen from European chestnut pollen--Cas s 1.

Pollens from trees of the order Fagales (e.g. birch, alder, hazel, and hornbeam) all contain one major allergen--the main cause for tree pollen allergy. So far the major allergens from birch (Bet v 1), alder (Aln g 1), hazel (Cor a 1), and hornbeam (Car b 1) have been characterized, showing high sequence similarity with each other (1-4). We present the molecular and immunologic characterization of Cas s 1, the major allergen from the European chestnut (Castanea sativa). From aqueous pollen extracts from European chestnut a protein was purified to homogeneity and was subjected to further investigation. The protein revealed a Mr of 22 kDa and was shown to represent the major allergen of the European chestnut (immunoblotting, histamine release) and designated Cas s 1. Despite a marked difference in Mr, Cas s 1 shows significant amino acid sequence similarity at the N-terminus and is antigenically closely related to the major birch pollen allergen Bet v 1 (17 kDa), as shown by binding to the anti-Bet v 1 monoclonal antibody BIP-1 and by IgE-inhibition tests using recombinant Bet v 1.

Allergens↗
PubMed1993

Four recombinant isoforms of Cor a I, the major allergen of hazel pollen, show different IgE-binding properties.

Previous studies showed that pollens from trees of the order Fagales (e.g. birch, alder, hazel and hornbeam) all contain one major allergen. These proteins are cross-reactive between these tree species, and approximately 95% of tree-pollen-allergic patients display IgE binding to these allergens. Using the reported N-terminal amino acid sequence of the hazel pollen allergen Cor a I, it was possible to amplify Cor-a-I cDNA by use of the polymerase chain reaction. Four clones with cDNA inserts were isolated. All four clones contained an open reading frame of 477 nucleotides (159 amino acids) but differed in length of their 3'-non-coding regions. Within the overlapping regions, the nucleotide sequence of the 3'-non-coding regions of the four clones were nearly identical. The open reading frames coded for different isoforms of the major hazel pollen allergen, Cor a I. The clones were designated Cor a I/5, 6, 11 and 16, respectively. Comparison of the deduced amino acid sequences of these Cor a I isoforms revealed identities of 96-99%. The sequence identities between the Cor a I isoforms and Bet v I, the major birch pollen allergen, were 71-73% (80.5-83% similarity). Comparing amino acid sequences of Cor a I isoforms with the published sequences of Aln g I, the major allergen from alder, and Car b I and isoforms, the major allergen from hornbeam, 75.5-76.7% identity (83.6-85% similarity) and 83.6-89.9% sequence identity (89.3-95% similarity), respectively, was found. The four Cor a I cDNAs were subcloned into plasmid pKK223-3 and expressed in Escherichia coli as non-fusion proteins; their capacity to bind serum IgE from tree-pollen-allergic patients was investigated. The four cloned isoforms showed an apparent molecular mass of 17 kDa in SDS/PAGE, identical to the natural, pollen-derived Cor a I. IgE antibodies from tree-pollen-allergic patients reacted with all four recombinant isoforms. However, we noted marked differences in the IgE-binding patterns of the distinct isoforms. Furthermore, Cor a I/11 was the only isoform recognized by the anti-(Bet v I) mAb, BIP 1. Our results demonstrate that Cor a I isoforms display different antigenic and allergenic properties, very likely due to few but significant changes in their amino acid sequences. These findings have implications for the development of reagents for diagnosis and immunotherapy of type I allergies.

Allergens↗
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

Multiple T cell specificities for Bet v I, the major birch pollen allergen, within single individuals. Studies using specific T cell clones and overlapping peptides.

Twenty-five T cell clones specific for Bet v I were established from the peripheral blood of two birch pollen-allergic patients. The T cell epitopes of these clones were mapped using dodecapeptides overlapping for 2 amino acids (neighbors share 10 residues) spanning the whole amino acid sequence of the protein (159 amino acids). In total, 7 epitopes could be detected. One donor displayed 6 distinct T cell specificities for the Bet v I molecule in 14 T cell clones; for the other donor, 4 stimulating peptides for 11 clones could be identified. Two T cell epitopes were recognized by both subjects. One of these might represent an immunodominant epitope located at amino acid position 77-92 of the Bet v I molecule, as in 13/25 T cell clones activation could be induced by this amino acid sequence. One T cell clone reacted with purified pollen-derived Bet v I, but neither with any peptide synthesized according to a Bet v I-encoding cDNA nor with the respective recombinant protein. Upon stimulation with allergen, the majority of the clones (21/24) revealed the TH0 or TH2 type of cytokine production (interleukin-4 production), indicating their importance in the pathogenesis of the allergic disease.

Allergens↗
PubMed1993

The expression and cellular distribution of adhesion molecules CD2/LFA-3 and ICAM-1/LFA-1 on mononuclear cells in squamous cell carcinoma of the head and neck.

The expressions and cellular distributions of two pairs of adhesion molecules CD2/LFA-3 (leukocyte function-associated antigen-3) and LFA-1/ICAM-1 (intercellular adhesion molecule-1) were examined in inflammatory cellular infiltrates of advanced squamous cell carcinomas of the head and neck by immunohistochemical techniques including double-staining methods. Thirteen patients were investigated using the following monoclonal antibodies (mAbs): CD2, LFA-3 (CD58), ICAM-1 (CD54), LFA-1 (CD11a), the alpha/beta and gamma/delta T-cell receptor, pan T cells and broadly distributed monocyte/macrophage (m/m phi) [Fc gamma RII (CD32), 25F9, RM3/1]. LFA-3 staining was observed on a high number of cells (968 +/- 112 cells/mm2), correlating to the number of Fc gamma RII (CD32; P < 0.01), 25F9 (P < 0.05) and RM3/1 (P < 0.05) positive m/m phi. Its ligand CD2 was found on 365 +/- 126 cells/mm2, representing about 50% of CD3+ cells (730 +/- 286 cells/mm2). CD2 positivity correlated to CD3 and CD8 (P < 0.01) but not to CD4+ T cells. LFA-1 and ICAM-1 were expressed on lymphocytes as well as on m/m phi. ICAM-1+ cells (902 +/- 205 cells/mm2) correlated to CD3+, CD8+ and RM3/1+ cells (P < 0.01). LFA-1 positivity (803 +/- 255 cells/mm2) showed correlations to nearly all investigated antigens, as well as to CD4+ T cells (P < 0.05). These results show that different m/m phi subsets display distinct patterns of adhesion molecule expressions suggesting different pathways of regulation. The CD3+ lymphocyte population revealed a lack of CD2 expression that was more pronounced in the CD4+ subset and indicated impaired lymphocyte function.

Adult↗
PubMed1993

Hydroxyethylstarch deposits in human skin--a model for pruritus?

Severe itching for unknown reasons has been reported after administration of hydroxyethylstarch (HES) in haemodilution therapy of humans. After HES treatment, vacuoles in cells of various organs in humans have been shown, predominantly affecting the mononuclear phagocyte system. These vacuoles present indirect evidence for phagocytosis of HES particles. Since phagocytosis is also known to occur in the skin, this organ might represent a target for HES deposition, resulting in subsequent release of mediators responsible for the observed itching. The aim of the present investigation was to study skin biopsies of patients, who had received HES and suffered subsequently from itch. Skin sections were investigated for morphological impairment by means of light and electron microscopy, immunohistochemistry and immunoelectron microscopy using a polyclonal anti-HES antiserum. Storage of HES was demonstrated in the skin of all patients, mainly in dermal macrophages, endothelial cells of blood and lymph vessels, some perineural cells and endoneural macrophages of larger nerve fascicles, some keratinocytes and Langerhans cells. Treatment with antihistaminic agents proved ineffective in these patients; this fits with the observation that morphological signs of histamine release from mast cells were absent. These findings indicate that other mediators from HES-affected cells must be responsible for the development of the itching. Thus, investigation of HES storage may be a useful contribution to the elucidation of release of itch mediators and induction of pruritus.

Adult↗
PubMed1993

T cell clones specific for Bet v I, the major birch pollen allergen, crossreact with the major allergens of hazel, Cor a I, and alder, Aln g I.

Tree pollens are responsible for type I allergies during the flowering season in spring. Pollens from birch, hazel and alder constitute the most important allergen sources in this respect in the northern hemisphere. Human IgE antibodies, specific for the major allergens of these pollens, are known to crossreact, and in general every tree pollen allergic patient is sensitized to these three pollen allergens. In this study we investigated eight T-helper cell clones (CD3+, CD4+, TCR alpha/beta) with specificity for Bet v I, the major birch pollen allergen, as proved by reactivity with purified natural as well as with recombinant allergen. The T cell clones were used to investigate common T cell epitopes of the Bet v I molecule with Cor a I, the major allergen of hazel pollen and Aln g I, the major allergen of alder pollen. All eight T cell clones reacted with all three proteins with different intensity. Moreover, three T cell clones, which were known to react with immunodominant T cell epitopes on the Bet v I molecule, were tested for reactivity with dodecapeptides synthesized according to the corresponding homologous regions of the Cor a I and Aln g I sequence. All the peptides induced strong T cell proliferation, indicating the existence of multiple cross-reacting epitopes. These findings will have an impact on the production of vaccines for immunotherapy of tree pollen allergies.

Allergens↗