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H Breiteneder

Publications and source records attributed to H Breiteneder.

At least 109 records · Page 6Linked to original sources
PubMed1994

Effects of 17 beta-estradiol on circulating adhesion molecules.

The effects of 17 beta-estradiol (E2) on the serum levels of the circulating endothelial-leukocyte, intercellular, and vascular adhesion molecules [ELAM-1, ICAM-1 (CD54), and VCAM-1] were evaluated in healthy male volunteers after single im injection of 10 mg E2 valerate. In addition, a time course of the effects of E2 on circulating adhesion molecules (AMs), cortisol serum levels, differential blood counts, and surface expression of the lymphocyte function-associated antigen-1 (CD11a/CD18), CD3, CD4, CD19, and CD25 on leukocytes was studied in another group of volunteers. A 5% decrease in circulating ICAM-1 (P = 0.045 vs. placebo) was found when a single time point (96 h after E2 injection) was studied. However, this decrease was smaller than the intrasubject (day to day) variability observed, and there was no consistent and time-dependent effect of E2 on circulating AMs. Circulating neutrophils increased 2.3-fold over baseline after E2 treatment (P = 0.0008 vs. placebo). The mean coefficients of variation for the intrasubject (day to day) and intersubject variability of circulating AMs were between 5.4-7.5% and 20-29%, respectively. Our findings indicate that the effect of E2 on circulating AMs is not distinguishable from the intrasubject variability observed after placebo treatment. Thus, an effect of E2 on adhesion molecules is unlikely to contribute to the antiatherogenic-cardioprotective effect of E2. The pronounced E2-mediated increase in neutrophils deserves further studies to elucidate its (patho-)physiological implications.

Adult↗
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↗
PubMed1993

Purification and characterization of recombinant Bet v I, the major birch pollen allergen. Immunological equivalence to natural Bet v I.

Pollen from trees of the order Fagales (e.g. birch, alder, hazel, oak, and hornbeam) are a major cause of Type I allergies observed in early spring. Previously, we reported the cloning and sequencing of Bet v I, the major birch pollen allergen, which showed high sequence similarities to a family of plant pathogen-activated genes (Breiteneder, H., Pettenburger, K., Bito, A., Valenta, R., Kraft, D., Rumpold, H., Scheiner, O., and Breitenbach, M. (1989) EMBO J. 8, 1935-1938). Here, we present the results on the expression, purification, and characterization of recombinant Bet v I produced in Escherichia coli as fusion and non-fusion protein, respectively. The purified recombinant proteins were analyzed to verify purity and structural integrity, and their immunological properties were compared to those of Bet v I isolated from birch pollen (natural Bet v I). Immunoblot analyses showed that the recombinant proteins are specifically recognized by monoclonal antibodies raised against natural Bet v I as well as by IgE from birch pollen-allergic patients. However, enzyme-linked immunosorbent assays revealed a decreased IgE-binding activity of the recombinant fusion Bet v I compared to the non-fusion and natural Bet v I proteins, which probably results from conformational changes due to the fusion tail. Recombinant non-fusion Bet v I was equivalent to natural Bet v I with respect to IgE-binding properties, the ability to induce in vitro proliferation of allergen-specific T-cell clones, and the ability to release histamine from basophils derived from birch pollen-allergic patients.

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

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↗
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↗
PubMed1992

Association between IgE response against Bet v I, the major allergen of birch pollen, and HLA-DRB alleles.

The association of the human IgE response against Bet v I, the major allergen of birch pollen, and the HLA-DR and DQ phenotype was studied. Birch pollen allergic patients showed a typical case history, positive skin-prick test, and positive RAST with birch pollen extracts. They were divided into two groups. Group I (n = 37) consisted of individuals generating IgE antibodies that selectively reacted with Bet v I. Their serum IgE did not react with minor allergens from birch pollen as tested by immunoblot analysis, nor did they show a response against allergens from a panel of grass and other tree pollen or perennial allergens from animals and fungi as determined by skin-prick test. Patients belonging to group II (n = 34) possessed IgE reacting with Bet v I plus one or more additional allergens. The control group consisted of 637 healthy blood donors. Comparison of the frequencies of RFLP-defined HLA-DR and DQ alleles in patients and the control group revealed that the distribution of DRB3 alleles in group I patients differed significantly from that in the control group: A higher frequency of the DRw52a/c alleles in comparison to the control group (pcorr less than 0.02) was observed. In addition, alleles defined by nucleotide sequences coding for the amino acid sequence tyrosine-phenylalanine-histidine at positions 30-32 of the beta chain of DR molecules were found with a higher frequency in patient group I (pcorr less than 0.02), too. These alleles comprise DRw52a/c and some DRB1 alleles.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗
PubMed1991

Homology of the major birch-pollen allergen, Bet v I, with the major pollen allergens of alder, hazel, and hornbeam at the nucleic acid level as determined by cross-hybridization.

To investigate the relationship of the major allergens of birch (Bet v I), alder (Aln g I), hazel (Cor a I), and hornbeam (Car b I) at the nucleic acid level, a cDNA clone coding for the complete Bet v I protein was used for Northern and Southern blot experiments. RNAs were isolated from pollen of birch (Betula verrucosa), alder (Alnus glutinosa), hazel (Corylus avellana), and hornbeam (Carpinus betulus). Hybridization was performed at different stringencies. At high stringency, comparable binding of the complete Bet v I cDNA probe to pollen RNAs from birch, alder, and hazel could be observed, indicating high homology of the mRNAs coding for these allergens. With the 3' and 5' half fragments of the Bet v I cDNA, both probes bound to transcripts of all four tree pollens, but most strongly to birch RNA. In Southern blots, distinct binding patterns of genomic DNA digests of birch, alder, hazel, and hornbeam were observed. Most bands were observed with birch DNA digests and less with alder, whereas in genomic DNA digests of hornbeam and hazel, only one band was observed. The result of these cross-hybridization experiments indicate a high homology at the nucleic acid level of the four major allergens of trees belonging to the order Fagales. The sequence similarity presented here further corroborates earlier observations of immunologic cross-reactivity at the protein level. Therefore, in the case of the major allergens of the Betulaceae, an extract with only one major allergen, preferentially Bet v I, instead of all four major allergens, should be sufficient for diagnostic and therapeutic purposes.

Allergens↗
PubMed1989

The gene coding for the major birch pollen allergen Betv1, is highly homologous to a pea disease resistance response gene.

Pollen of the white birch (Betula verrucosa) is one of the main causes of Type I allergic reactions (allergic rhinoconjunctivitis, allergic bronchial asthma) in Middle and Northern Europe, North America and the USSR. Type I allergies are a major threat to public health in these countries, since 10-15% of the population suffer from these diseases. BetvI, an allergenic protein with an Mr of 17 kd is a constituent of the pollen of white birch and is responsible for IgE binding in more than 95% of birch pollen allergic patients. Here, we report the complete nucleotide sequence and deduced amino acid sequence of a cDNA clone coding for the major pollen allergen (BetvI) of white birch. It is similar to the N-terminal peptide sequences of the allergens of hazel, alder and hornbeam (close relatives) but it has no significant sequence homology to any other known allergens. However, it shows 55% sequence identity with a pea disease resistance response gene, indicating that BetvI may be involved in pathogen resistance of pollen.

Allergens↗
PubMed1988

Physical map and protein gene map of cyanelle DNA from the second known isolate of Cyanophora paradoxa (Kies-strain).

A restriction map of the cyanelle DNA from a different isolate of Cyanophora paradoxa (Kies-strain) was established. The positions of 18 protein genes and the rRNA genes have been located and compared to the positions of these genes from the first isolate of C. paradoxa (Pringsheim-strain). The gene arrangement is absolutely conserved in both cyanelle DNAs. The differences in size (ca. 9 kb) and the unrelatedness in the restriction patterns could be explained by numerous small insertions into intergenic regions of the cyanelle chromosomes.

Cyanobacteria↗
PubMed1988

Isolation and characterization of messenger RNA from male inflorescences and pollen of the white birch (Betula verrucosa).

A glycoprotein with a molecular weight (MW) of 17 kilodaltons (kD), Bet v I, represents the major allergen of the white birch (Betula verrucosa, BV) and plays an important role in tree-pollen-induced type I allergic reactions. In order to characterize the major and also some minor allergens of BV, we investigated the IgE-binding properties of these allergens using immunoblot techniques. Normal and patients' sera were employed for this study. Furthermore, RNA from male inflorescences and from pollen of BV were isolated and purified by affinity chromatography on oligo-dT-cellulose. Poly(A)+-mRNA thus obtained was translated in vitro in a cell-free wheat germ system and the proteins synthesized were separated by SDS-PAGE and transferred to nitrocellulose. The blots were incubated with normal human sera and with sera from patients allergic to birch pollen. Bound IgE antibodies were detected with 125I-labeled anti-IgE. We observed major IgE binding to a protein of an MW of 12.5 kD, and little IgE binding to a 17-kD protein, presumably Bet v I. Comparing the products of in vitro translation from mRNA preparations of mature pollen and of male inflorescences collected in June, October and February, little seasonal variations could be observed. As the in vitro translation system does not glycosylate proteins, our results show that the majority of IgE in patients' sera is not directed against the carbohydrate moieties of these allergens.

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↗
PubMedDate not supplied

Cloning of the patatin-like latex allergen Hev b 7, its expression in the yeast Pichia pastoris and its immunological characterization.

The 43-kD latex allergen Hev b 7 was purified from the latex of Hevea brasiliensis and identified by N-terminal and internal peptide sequences as highly homologous to patatins. Patatins are storage proteins encoded by a multigene family found in plants such as potato and tomato. We have obtained a cDNA clone coding for a cytoplasmic form of Hev b 7. The recombinant protein was expressed in the methylotrophic yeast Pichia pastoris at 10 mg/l culture supernatant. Both natural Hev b 7 and rHev b 7 were recognized by IgE in 11% of the latex-allergic patients. rHev b 7 inhibited binding to its counterpart in natural rubber latex extracts. Purified rHev b 7 used at concentrations of 10 micrograms/ml in skin prick tests produced wheal-and-flare reactions of sizes equal to those produced by nHev b 7. Furthermore, we were able to show that rHev b 7 possessed esterase activity. A plant expression system for the production of larger quantities of recombinant latex allergens as an alternative to the preparation from H. brasiliensis sap is discussed.

Allergens↗
PubMedDate not supplied

Rapid production of recombinant allergens in Nicotiana benthamiana and their impact on diagnosis and therapy.

BACKGROUND: Type I allergies are immunological disorders that afflict a quarter of the world's population. Recombinant allergens have improved the diagnosis of allergic diseases and allow the formulation of new therapeutic approaches. Over 50% of all allergens are of plant origin. OBJECTIVE: We have applied a novel method of overexpressing plant allergens in the tobacco-related species Nicotiana benthamiana. METHOD: This method is based on the use of a chimeric tobacco mosaic virus that harbors a foreign gene sequence and directs its transcription after the infection of the host plant. RESULTS: We have expressed the model allergen Bet v 1, the major birch pollen allergen, and two Hevea brasiliensis latex allergens, the spina-bifida-associated allergens Hev b 1 and Hev b 3, in N. benthamiana using such a viral vector. Bet v 1, Hev b 1 and Hev b 3 produced by this method were recognized by patients' IgE suggesting that the plant-produced allergens were properly folded. Nonpurified Bet v 1 expressed in N. benthamiana leaves had the same immunogenicity as purified Bet v 1 expressed in Escherichia coli or natural Bet v 1 when tested in a murine model of type I allergy. CONCLUSION: We conclude that this plant expression system offers a viable alternative to fermentation-based production of allergens in bacteria or yeasts.

Allergens↗
PubMedDate not supplied

IgE mimotopes of birch pollen allergen Bet v 1 induce blocking IgG in mice.

BACKGROUND: The induction of nonanaphylactogenic 'blocking' IgG antibodies capable of inhibiting the IgE/allergen interaction represents a favorable therapeutic concept for type I allergy. However, IgG antibodies to allergens may block or enhance specific IgE binding, depending on the recognized epitope. Taking the major birch pollen allergen Bet v 1 as a model, we developed a strategy for the precise induction of IgG antibodies of a desired epitope specificity. METHODS: Random phage display peptide libraries were applied to define peptide structures mimicking natural epitopes (mimotopes) of Bet v 1. Selections were performed with BIP 1, a murine monoclonal antibody known to enhance the IgE binding to Bet v 1, and with anti-Bet v 1 IgE purified from patients' sera. The characterized Bet v 1 mimotopes were used to localize the corresponding epitope at the surface of Bet v 1 by a computer-aided mathematical approach based on the three-dimensional structure and the chemical character of the amino acids. The Bet v 1 mimotopes were further used to immunize BALB/c mice. The specificity of the induced antibodies was tested by immunoblotting and inhibition assays. RESULTS: With the three-dimensional epitope search it became possible to localize a discontinuous IgE epitope on the surface of Bet v 1 in a substantial distance from the IgG epitope of the monoclonal antibody BIP 1. Moreover, we could demonstrate that phage displaying mimotopes are immunogenic vectors for the precise induction of epitope-specific IgG. Immunization with BIP 1 mimotopes induced IgG enhancing the IgE binding to Bet v 1, whereas immunization with IgE mimotopes resulted in IgG capable of blocking human IgE binding in vitro. CONCLUSION: Allergen mimotopes can be used for the induction of anti allergen IgG of desired specificity. We propose that mimotope immunotherapy based on IgE mimotopes generated by biopannings may represent a future concept for therapy of type I allergy.

Allergens↗