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Biomedical subjects

O Scheiner

Publications and source records attributed to O Scheiner.

At least 109 records · Page 6Linked to original sources
PubMed1995

The basic isoform of profilin in pathogenic Entamoeba histolytica. cDNA cloning, heterologous expression, and actin-binding properties.

In the human parasite Entamoeba histolytica, components of the cytoskeleton are involved in the pathogenicity by their contribution to immune evasion by antibody capping and shedding. In this study, we focus on profilin as a central regulatory component of the cytoskeleton. Profilin was isolated from trophozoites of the pathogenic E. histolytica strain SFL-3, and partial amino acid sequences were used to devise a probe for isolating a profilin cDNA. The deduced complete primary structure was divergent: plant profilins with amino acid sequence identities in the range 33-38% were more closely related than the mammalian profilins with sequence identities 21-28%. The cDNA was expressed as a nonfusion protein in Escherichia coli. Isoelectric focussing of the natural profilin isolated from E. histolytica showed two isoforms with different isoelectric points; the recombinant profilin migrated with the basic isoform. In a blot overlay experiment, purified 125I-labeled recombinant profilin bound not only to plant actin, but also to mammalian actin, demonstrating that cytoskeletal components from distantly related organisms with divergent primary structures can be compatible.

Actins↗
PubMed1995

Molecular characterization of Api g 1, the major allergen of celery (Apium graveolens), and its immunological and structural relationships to a group of 17-kDa tree pollen allergens.

Individuals suffering from immediate hypersensitivity (type-I allergy) to a particular pollen frequently display intolerance to several foods of plant origin. In this respect, individuals sensitized to birch pollen and/or mugwort pollen frequently display type-I allergic symptoms after ingestion of celery. In this study, we expressed the major allergenic protein of celery, Api g 1, which is responsible for the birch-celery syndrome, in the form of a non-fusion protein. The open reading frame of the cDNA of Api g 1 codes for a protein of 153 amino acids with a molecular mass of 16.2 kDa and 40% identity (60% similarity) to the major allergen of birch pollen, Bet v 1. Furthermore, Api g 1 exhibited similar characteristics to (a) two proteins in parsley induced by fungal infection, (b) the major tree pollen allergens and (c) pathogenesis-related and stress-induced proteins in other plant species. The reactivity of recombinant Api g 1 with IgE antibodies present in sera from celery intolerant patients was comparable to that of the natural celery allergen. Cross-reactivity with Bet v 1 was proven by cross-inhibition experiments, which provides further support for the existence of the birch-celery syndrome and for the suggestion that allergies to some vegetable foods are epiphenomena to allergies caused by inhalation of tree pollen.

Allergens↗
PubMed1995

High level expression of birch pollen profilin (Bet v 2) in Escherichia coli: purification and characterization of the recombinant allergen.

Up to 20% of the population in industrialized countries suffer from type I allergic symptoms (rhinitis, conjunctivitis, and bronchial asthma). The cDNA coding for birch pollen profilin, a highly conserved cross-reactive allergen and actin-binding protein was expressed in Escherichia coli. Upon induction with IPTG up to 30 mg recombinant profilin per liter culture could be obtained. A single step purification protocol based on the high affinity of profilin to poly-(L-proline) Sepharose was used to obtain large amounts of soluble and pure recombinant birch profilin. Recombinant birch pollen profilin specifically bound IgE, elicited dose dependent histamine release from patients basophils and could be used for skin prick testing without toxic effects. The results indicate that by using purified recombinant profilin, specific diagnosis of type I allergy might be improved.

Adsorption↗
PubMed1995

Cloning and sequencing of Mal d 1, the major allergen from apple (Malus domestica), and its immunological relationship to Bet v 1, the major birch pollen allergen.

The number of tree-pollen-allergic patients showing allergic reactions to apples, various vegetables and nuts is increasing. In this paper the molecular characterization of the major apple allergen, Mal d 1, is reported. The cDNA coding for Mal d 1 was cloned and sequenced. Its open reading frame codes for a protein of 159 amino acids with a predicted molecular mass of 17.7 kDa and a predicted pI of 5.9. Sequence comparison to Bet v 1, the major birch pollen allergen, revealed 64.5% identity on the amino acid level and 55.6% identity on the nucleic acid level. Recombinant Mal d 1 was expressed in the plasmid pMW 175 in E. coli BL 21 (DE3) and its immunological properties were tested. Crossreactivity with Bet v 1 was shown by inhibition assays.

Allergens↗
PubMed1995

Purification of human basophils and mast cells by multistep separation technique and mAb to CDw17 and CD117/c-kit.

Basophils and mast cells represent distinct cell lineages within the hemopoietic system. Based on the unique cell surface antigen profile of both cells, we have established methods which allow the reproducible purification to homogeneity (> 99%) of normal human basophil granulocytes from the peripheral blood and of mast cells from human dispersed tissues. Basophils (n = 9) were purified by current counterflow elutriation followed by depletion of monocytes with CD14 mAb conjugated to magnetic beads, and subsequent cell sorting for CD217+ cells. Basophil purity was 99.5 +/- 0.4% (range 98.7-99.9%). Mast cells were obtained from lung (n = 6), uterus (n = 1), mastocytosis bone marrow (n = 2), and human foreskin (n = 2). Mast cells were purified by collagenase digestion followed by current counterflow elutriation and sorting with CD117/c-kit mAb. Mast cell purity was 99.4 +/- 0.7% (range: 97.5-99.9%). Purified cells were more than 90% viable and were able to release histamine on induction with IgE plus anti-IgE. Furthermore, the PCR technique could be applied on pure cells and confirmed expression of high affinity IgE receptor (Fc epsilon R1) alpha chain mRNA. Thus, by combining isolation techniques including elutriation, magnetic cell depletion and cell sorting with mAb, functionally intact normal human basophils and mast cells can be enriched to homogeneity.

Antibodies, Monoclonal↗
PubMed1995

Nonallergic individuals recognize the same T cell epitopes of Bet v 1, the major birch pollen allergen, as atopic patients.

The immune response toward allergens in nonallergic healthy individuals was investigated. To boost immune responses, two injections of birch pollen extract were administered to five nonallergic volunteers. T cell lines (TCL) with specificity for Bet v 1, the major birch pollen allergen, were established and analyzed for epitope specificity using overlapping peptides. Forty-nine T cell clones (TCC) specific for Bet v 1 were isolated from TCLs. Comparison with TCL and TCC established from birch pollen-allergic patients was performed. All TCC revealed the Th phenotype. Epitope specificities of TCL and TCC from nonatopics were identical to those found in allergic individuals. No association between MHC class II molecules and particular epitopes could be observed. In nonallergic as well as in allergic individuals, cytokine production in response to specific stimulation revealed a majority of Th-clones producing IL-4 and IFN-gamma. However, TCC derived from atopic individuals revealed a higher IL-4/IFN-gamma ratio. Immunoblot and ELISA revealed Bet v 1-specific IgG in nonallergic individuals before and after booster injections, but no IgE could be detected. High levels of Bet v 1-specific IgG and IgE could be detected in birch pollen-allergic patients. It can be concluded, that nonatopic and allergic individuals display the same repertoire of T cell specificities. In allergic individuals, the activation of allergen-specific TCC leads to a higher ratio of produced IL-4 vs IFN-gamma, which is responsible for enhanced IgE production.

Allergens↗
PubMed1995

Isoforms of Bet v 1, the major birch pollen allergen, analyzed by liquid chromatography, mass spectrometry, and cDNA cloning.

Bet v 1, the major allergen of birch pollen, displays a considerable degree of heterogeneity. Several charge variants have been detected by two-dimensional IgE immunoblots and isoelectric focusing techniques. This heterogeneity has been attributed to glycosylation (or other post-translational modifications) or to isogenes coding for Bet v 1 isoforms and/or allelic variants. However, until now, only limited structural data for Bet v 1 have been published. Recently, we described the expression, purification, and immunological properties of recombinant Bet v 1 (rBet v 1) produced in Escherichia coli as a non-fusion protein (Ferreira, F. D., Hoffmann-Sommergruber, K., Breiteneder, H., Pettenburger, K., Ebner, C., Sommergruber, W., Steiner, R., Bohle, B., Sperr, W. R., Valent, P., Kungl, A. J., Breitenbach, M., Kraft, D., and Scheiner, O. (1993) J. Biol. Chem. 268, 19574-19580). Here, we present a more detailed structural characterization of Bet v 1 by both cDNA cloning and mass spectrometry. Thirteen different cDNA clones coding for Bet v 1 isoforms were obtained by polymerase chain reaction amplification of birch pollen cDNA with a sequence-specific 5'-terminal primer and a nonspecific 3'-terminal primer or by immunological screening of a birch pollen cDNA library. These isoforms are referred to as Bet v 1b to Bet v 1n, whereas the previously isolated Bet v 1 cDNA (Breiteneder, H., Pettenburger, K., Bito, A., Valenta, R., Kraft, D., Rumpold, H., Scheiner, O., and Breitenbach, M. (1989) EMBO J. 8, 1935-1938) is now referred to as Bet v 1a. High performance liquid chromatography and plasma desorption mass spectrometry of proteolytic fragments of purified natural Bet v 1 (nBet v 1) and rBet v 1a were used to (i) confirm the primary structure of all Bet v 1 isoforms and (ii) to investigate any possible postsynthetic modifications on rBet v 1a or on the natural mixture of isoallergens obtained from birch pollen. Except for the cleavage of initiating methionine, no postsynthetic modifications were found in either nBet v 1 or rBet v 1a.

Allergens↗
PubMed1995

Tumor necrosis factor-alpha induction of major histocompatibility complex class II antigen expression is inhibited by interferon-gamma in a monocytic cell line.

Regulation of major histocompatibility complex (MHC) class II antigen expression by cytokines has been suggested to play a major role in the initiation and propagation of immune and autoimmune processes. The analysis of class II gene regulation benefits greatly from the existence of mutants with defects in regulatory factors. We report the establishment of a subclone of the human monocytic cell line U937, termed C119/9, with unusual cytokine regulation of MHC class II expression. In contrast to the parental U937 cell line, only tumor necrosis factor (TNF)-alpha, and not interferon (IFN)-gamma induces the expression of MHC class II antigens on C119/9 cells, and paradoxically, this induction was inhibited almost completely by IFN-gamma. The HLA-DR induction is controlled at the transcriptional level by the first 150 bp of the class II promoter which contains all the class II consensus elements. Both HLA-DR and -DQ mRNA are induced by TNF-alpha treatment, and both are diminished upon co-treatment with TNF-alpha and IFN-gamma. This antagonism between TNF-alpha and IFN-gamma seem to be restricted to MHC class II genes. This subline of U937 cells may be useful in further studies of MHC class II regulation.

Blotting, Northern↗
PubMed1995

Diversity of TCRAV and TCRBV sequences used by human T-cell clones specific for a minimal epitope of Bet v 1, the major birch pollen allergen.

T-cell clones (TCC) were raised from the peripheral blood of patients suffering from tree pollen allergy. All TCC were restricted by HLA-DR molecules. In order to investigate possible intervention targets in Type I allergic diseases, we examined T-cell receptor (TCR) alpha and beta chain nucleotide sequences of five allergen-reactive human CD4+ TCC specific for a C-terminal epitope (BV 144) of Bet v 1, the major birch pollen allergen. Proliferation assays using synthetic peptides revealed the 10-mer LRAVESYLLA as minimal epitope for three TCC; two TCC also displayed reactivity with the nonapeptide LRAVESYLL. Two TCC expressed TCRBV2S3, all other BV144-specific TCC used diverse TCRAV and TCRBV gene segments. Moreover, the junctional regions encoding the third complementary determining regions (CDR3) of the TCR showed a striking heterogeneity in length and amino acid composition. Nevertheless, all TCC showed an arginine residue in the N-terminal region of their TCRBV CDR3 loops. Therefore, therapeutical strategies aimed at the clonal deletion of allergen-specific T-cell clones, providing help for IgE synthesis, will not be feasible. Our results cast a doubt on the theory that the CDR3 exclusively provides the primary contact with the peptide bound in the major histocompatibility (MHC) groove, and suggest additional interaction with MHC class II.

Allergens↗
PubMed1995

Molecular analysis of two hexokinase isoenzymes from Entamoeba histolytica.

The zymodemes, electrophoretic patterns of hexokinase, phosphoglucomutase and glucose phosphate isomerase isoenzymes, have been widely used to determine the pathogenicity of Entamoeba histolytica isolates. Although pathogenic and nonpathogenic forms of E. histolytica differ clearly in sequences of many homologous genes, a conversion between pathogenic and nonpathogenic zymodemes has been reported by several laboratories. To approach the question what might be the basis for the observed conversion, we examined the molecular biology of the hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) isoenzymes in pathogenic E. histolytica. We isolated two different cDNAs pHXK1 and pHXK2 coding for polypeptides with significant sequence similarity to hexokinases and deduced molecular masses of 49.8 kDa and 49.4 kDa. The two hexokinase sequences differed by 11% on the amino acid and by 8% on the nucleotide level. Expression of the cDNAs in Escherichia coli as nonfusion proteins gave two polypeptides with hexokinase activity. The recombinant Hxk1 and Hxk2 polypeptides comigrated with the more basic and more acidic isoforms of pathogenic amoebae in starch gel electrophoresis, as well as in low and high resolution isoelectric focussing gels. This identified the observed hexokinase isoenzymes of pathogenic E. histolytica as the products of two genes, hxk1 and hxk2.

Amino Acid Sequence↗
PubMed1995

Allergen-specific IgE production of committed B cells from allergic patients in vitro.

The allergen-specific in vitro IgE synthesis in blood leukocytes from patients with allergy was monitored outside the pollen season with recombinant and natural pollen allergens and was compared with the total IgE production. The addition of interleukin-4 (IL-4) and antibody to CD40 increased the amount of total IgE by up to 20-fold in the culture supernatants of peripheral blood leukocytes from patients with allergy that could be antagonized by a neutralizing anti-IL-4 antibody in a dose-dependent manner. In contrast to total IgE, the amount of allergen-specific IgE was not affected by IL-4, and anti-CD40 or anti-IL-4, treatment. With oligonucleotides specific for IgE, complementary DNA from the amino terminal of the IgE heavy chain could be reversely transcribed and amplified by polymerase chain reaction from RNA of patients' unstimulated blood leukocytes, indicating that the IgE secretion in the cultures is due to a de novo IgE synthesis. It is concluded that the peripheral blood of patients with allergy contains long-lived allergen-specific B cells, which are not responsive to IL-4-mediated signals. These results may have implications for attempts to modulate specific IgE production in allergic patients with cytokines or cytokine antagonists.

Allergens↗
PubMed1995

Identification of allergens in fruits and vegetables: IgE cross-reactivities with the important birch pollen allergens Bet v 1 and Bet v 2 (birch profilin).

BACKGROUND: In this study serum samples collected from 20 patients with birch pollen allergy were investigated. All patients had experienced allergic symptoms after contact with or ingestion of particular fresh fruits and vegetables known as birch pollen-related foods. METHODS: Serum samples were tested by means of immunoblotting for IgE reactivities with proteins in extracts of birch pollen, apple, pear, celery, carrot, and potato. Anti-Bet v 1 and anti-Bet v 2 antibodies were used to investigate cross-reactivity. Inhibition studies were performed by preincubation of sera with recombinant Bet v 1 and Bet v 2. RESULTS: IgE binding to proteins, corresponding to the major birch pollen allergen Bet v 1 and to Bet v 2 (birch pollen profilin) could be observed. An allergen homologous to Bet v 1 could be detected in apple, pear, and celery when a Bet v 1-specific monoclonal antibody was used. Testing a polyclonal rabbit anti-Bet v 2 antibody with extracts of the respective plants revealed the presence of profilins in every source tested. Inhibition with recombinant Bet v 1 and Bet v 2 led to complete blocking or marked reduction of IgE binding to proteins of comparable molecular weights in the respective food extracts, indicating IgE cross-reactivity. CONCLUSION: Our results indicate that many plant-derived food agents contain proteins with high homology to the birch pollen allergens Bet v 1 and Bet v 2 and must therefore be considered as potentially threatening for patients with tree pollen allergy.

Allergens↗
PubMed1995

T-cell epitopes of Phl p 1, major pollen allergen of timothy grass (Phleum pratense): evidence for crossreacting and non-crossreacting T-cell epitopes within grass group I allergens.

BACKGROUND: The use of peptides representing T-cell epitopes of allergens is a modern concept for improvement of specific immunotherapy. A prerequisite for this approach is the identification of T-cell epitopes of atopic allergens. METHODS: T-cell lines and 40 T-cell clones (TCC) specific for Phl p 1, the group I allergen of timothy grass (Phleum pratense), were established from the peripheral blood of nine patients allergic to grass pollen and mapped for epitope specificity by using overlapping dodecapeptides. Phenotype and cytokine production profile of TCC were investigated. Representative TCC were analyzed for HLA-restriction, T-cell receptor V beta gene usage, and crossreactivity with grass pollen extracts from Dactylis glomerata, Poa pratensis, Lolium perenne, Secale cereale, and selected amino acid sequence-derived peptides. RESULTS: Patients displayed IgE binding to all grass species investigated. Forty TCC were established. Fifteen T-cell epitopes could be identified on Phl p 1. Of 40 TCC, 39 displayed the helper cell (Th) phenotype; one clone was CD8+. Specific stimulation induced a Th2-like type of cytokine production in 20 of 39 TCC. Crossreactivity studies revealed crossreacting and non-crossreacting T-cell epitopes. CONCLUSION: Phl p 1, a major grass pollen allergen, harbors multiple T-cell epitopes. Species-specific and crossreacting T-cell epitopes exist among group I allergens of grasses. Epitope recognition patterns could not be correlated with particular HLA haplotypes. A restricted T-cell receptor V beta gene usage was not observed.

Allergens↗
PubMed1995

Hydrocortisone enhances total IgE levels--but not the synthesis of allergen-specific IgE--in a monocyte-dependent manner.

Recently, hydrocortisone (HC), when combined with human IL-4, has been reported to increase IgE levels in supernatants (SN) of in vitro cultured leucocytes. In this study we investigated the influence of HC on allergen-specific IgE synthesis. Moreover, we examined the relevance of different cell types in this respect. Peripheral blood mononuclear cells (PBMC), T-cell depleted PBMC, CD14-depleted PBMC and highly purified B cells from 10 allergic (birch pollen and/or grass pollen) patients and five non-allergic individuals were investigated. The cells were incubated with HC and/or recombinant human IL-4 (rIL-4) for 8 days. A considerable increase of total IgE was observed in HC/rIL-4-stimulated cultures compared with rIL-4 alone, HC alone or non-stimulated cultures. We demonstrate that this effect depends on the presence of monocytes in in vitro cultures. These results were seen in every experiment, irrespective of healthy or atopic state of the blood donor. The increase of IgE could not be attributed to a rise of birch pollen-and/or grass pollen-specific IgE in patients allergic to these allergens, as shown by IgE-immunoblot. Radio-allergosorbent test (RAST) investigations of HC/rIL-4-stimulated cells cultures from allergic and non-allergic patients confirmed that HC/rIL-4-induced elevated IgE production was also not due to increased production of IgE, specific for important aero-allergens (pollens, house dust mite or animal dander). Therefore we conclude that newly synthesized IgE is not specific for allergens, but that sequential isotype switching in human B cells leads to increased polyclonal IgE production.

Allergens↗
PubMed1994

A birch gene family encoding pollen allergens and pathogenesis-related proteins.

Bet v I, the major pollen allergen of birch (Betula verrucosa), shows high sequence homology to a family of pathogenesis-related (PR) proteins that have recently been identified in several other plant species. We have used a pollen Bet v I cDNA clone and anti-Bet v I antibodies as probes to study the expression of Bet v I genes in birch cell suspension cultures under different experimental conditions. Induction of Bet v I-related proteins was detected in immunoblots of cell extracts upon co-cultivation with microbial pathogens. Northern analysis revealed the rapid induction of Bet v I transcripts in the presence of bacteria and fungi, but not by stress treatments (heat shock, metal ions) or by chemical elicitors. RNase protection experiments showed that the pathogen-inducible RNAs did not correspond to the pollen cDNA clone but most likely to the products of transcription of other members of the Bet v I gene family, sharing high sequence homology with the pollen-specific gene within the 5'-half of the coding region. We conclude that the Bet v I gene family of pollen allergens includes a subset of defense-related genes that are transcriptionally activated in the presence of microbial pathogens.

Allergens↗
PubMed1994

Four recombinant isoforms of Cor a 1, the major allergen of hazel pollen, show different reactivities with allergen-specific T-lymphocyte clones.

Purified preparations of allergenic proteins from plants, and in particular from pollens, consist of multiple closely related isoforms. These isoforms are highly similar in their amino acid sequences, yet they display different properties with respect to antibody binding. In this study we report of differential potencies of cross-reacting tree pollen allergens and cloned isoforms of these allergens to activate allergen-specific T-lymphocyte clones (T-cell clones; TCC). Six TCC with specificity for Bet v 1, a representative tree pollen major allergen, were established from peripheral blood of five birch-pollen-allergic donors. All TCC displayed the helper-cell phenotype. Five TCC reacted with distinct epitopes present on natural (n) and on recombinant (r) Bet v 1. One TCC could not be stimulated with r Bet v 1, in spite of strong reactivity with purified natural Bet v 1. The TCC were tested in proliferation assays using purified n Bet v 1, n Cor a 1 (the homologous major allergen of hazel pollen), r Bet v 1, four recombinant isoforms of Cor a 1 and peptides representing corresponding T-cell stimulating regions (isoepitopes) on these proteins. The clones showed different patterns of reactivity in response to stimulation with the five recombinant molecules and the corresponding peptides. Certain exchanges of amino acids within stimulating peptides correlated with a lack of proliferation of the TCC tested. These findings are important with respect to the use of broadly cross-reactive recombinant allergens or allergen-derived peptides for immunotherapy of type I allergy.

Allergens↗