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Biomedical subjects

O Scheiner

Publications and source records attributed to O Scheiner.

At least 91 records · Page 5Linked to original sources
PubMed1996

Dissection of immunoglobulin E and T lymphocyte reactivity of isoforms of the major birch pollen allergen Bet v 1: potential use of hypoallergenic isoforms for immunotherapy.

We dissected the T cell activation potency and the immunoglobulin (Ig) E-binding properties (allergenicity) of nine isoforms of Bet v 1 (Bet v 1a-Bet v 1l), the major birch pollen allergen. Immunoblot experiments showed that Bet v 1 isoforms differ in their ability to bind IgE from birch pollen-allergic patients. All patients tested displayed similar IgE-binding patterns toward each particular isoform. Based on these experiments, we grouped Bet v 1 isoforms in three classes: molecules with high IgE-binding activity (isoforms a, e, and j), intermediate IgE-binding (isoforms b, c, and f), and low/no IgE-binding activity (isoforms d, g, and 1). Bet v 1a, a recombinant isoform selected from a cDNA expression library using IgE immunoscreening exhibited the highest IgE-binding activity. Isoforms a, b, d, e, and 1 were chosen as representatives from the three classes for experimentation. The potency of each isoallergen to activate T lymphocytes from birch pollen-allergic patients was assayed using peripheral blood mononuclear cells, allergen-specific T cell lines, and peptide-mapped allergen-specific T cell clones. Among the patients, some displayed a broad range of T cell-recognition patterns for Bet v 1 isoforms whereas others seemed to be restricted to particular isoforms. In spite of this variability, the highest scores for T cell proliferative responses were observed with isoform d (low IgE binder), followed by b, 1, e, and a. In vivo (skin prick) tests showed that the potency of isoforms d and 1 to induce typical urticarial type 1 reactions in Bet v 1-allergic individuals was significantly lower than for isoforms a, b, and e. Taken together, our results indicate that hypoallergenic Bet v 1 isoforms are potent activators of allergen-specific T lymphocytes, and Bet v 1 isoforms with high in vitro IgE-binding activity and in vivo allergenicity can display low T cell antigenicity. Based on these findings, we propose a novel approach for immunotherapy of type I allergies: a treatment with high doses of hypoallergenic isoforms or recombinant variants of atopic allergens. We proceed on the assumption that this measure would modulate the quality of the T helper cell response to allergens in vivo. The therapy form would additionally implicate a reduced risk of anaphylactic side effects.

Allergens↗
PubMed1996

Toward selective elicitation of TH1-controlled vaccination responses: vaccine applications of bacterial surface layer proteins.

Bacterial surface layer proteins have been utilized as combined vaccine carrier/adjuvants and offer a number of advantages in these applications. The crystalline protein arrays contain functional groups in precisely defined orientations for coupling of haptens. Conventional applications of S-layer vaccines do not cause observable trauma or side effects. Depending on the nature of the S-layer preparations, antigenic conjugates will induce immune responses of a predominantly cellular or predominantly humoral nature. Immune responses to S-layer-hapten conjugates are also observed following oral/nasal application. In the present contribution, the status of investigations with S-layer conjugates in three main immunological projects is reviewed. In a project aimed at immunotherapy of cancer, conjugates of S-layer with small, tumor-associated oligosaccharides have been found to elicit hapten-specific DTH responses. An enlarged program of chemical synthesis has now been initiated to prepare a complete set of mucin-derived, tumor-associated oligosaccharides and their chemically modified analogues for elicitation of cell-mediated immune responses to certain tumors in humans. In another application, oligosaccharides derived from capsules of Streptococcus pneumoniae type 8 have been linked to S-layer proteins and have been found to elicit protective antibody responses in animals. Most recently, allergen S-layer conjugates have been prepared with the intention to suppress the TH2-directed, IgE-mediated allergic responses to Bet nu 1, the major allergen of birch pollen. In the former two applications, the S-layer vaccine technology appears to offer the versatility needed to direct vaccination responses toward predominant control by TH1 or TH2 lymphocytes to meet the different therapeutic or prophylactic requirements in each case. In the third application, work has progressed to a preliminary stage only.

Adjuvants, Immunologic↗
PubMed1996

Biological and immunological importance of Bet v 1 isoforms.

In 2D-PAGE analysis of Bet v 1, the major birch pollen allergen, up to 12 isoforms can be demonstrated that differ in their isoelectric points from about pH 4.9 to pH 5.9. The molecular weights of these isoforms seem to be rather similar, but minor variations can also be seen. Preliminary experiments with birch leaves seem to indicate that in aging leaves some isoforms can be found that do not occur in pollen. In birch cells cultured in vitro, Bet v 1 isoforms can be induced by bacterial infection that do not occur in pollen (Swoboda et al. (1995), Pant, Cell and Environment 18, 865-874). In a recent paper (Swoboda et al (1995)., J. Biol. Chem. 270, 2607-2613) we show that in natural Bet v 1 from pollen the isoforms are due to different protein sequences. The derived protein sequences of 10 different isoforms (corresponding to 13 different cDNAs) were determined and confirmed by plasma desorption mass spectrometry of purified natural Bet v 1 after trypsin and endoproteinase Glu-C digestion. These experiments also showed that pollen Bet v 1 isoforms were reactive to patients' sera to different degrees and that common post-synthetic modifications (besides N-terminal methionine cleavage) did not occur on Bet v 1. Recombinant isoforms were produced in E. coli, purified and tested with selected patients allergic to birch pollen (Ferreira et al., J. Exp. Med., in the press). The pattern of IgE binding to Bet v 1 isoforms widely differs. Also, T-cell clones from individual patients in some cases are specific to peptides occurring only in certain isoforms. It was of particular interest that three of the naturally occurring pollen Bet v 1 isoforms do not or hardly bind IgE of untreated patients allergic to Bet v 1. However, a comparison of IgE reactivity in patients before and after conventional immunotherapy with natural pollen extract clearly showed that this form of immunotherapy induced IgE to the isoforms that had been unreactive in untreated patients. One of these, Bet v 1d, showed a particularly strong potency towards T-cell stimulation. The isoform(s) that do not bind IgE in untreated patients but still show T-cell reactivity could be potentially utilized for a new form of immunotherapy that avoids the risk of anaphylaxis.

Allergens↗
PubMed1996

Diversity of human T cell receptor sequences of T cell clones with specificity for Bet v 1 peptide/MHC II complexes.

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 have examined T cell receptor (TCR) alpha- and beta-chain nucleotide sequences of several allergen-reactive human CD4+ TCC specific for four frequently found epitopes of Bet v 1, the major birch pollen allergen. In general, TCC specific for the 4 epitopes investigated, used diverse TCRAV and TCRBV gene segments. Moreover, the junctional regions encoding the third complementarity determining regions (CDR3) of the TCR showed striking heterogeneities in length and amino acid composition. A more restricted use of two J gene segments (TCRBJ1S4 and 2S7) was only observed in the beta-chain of TCR used by TCC specific for epitope 1. In addition, all TCC specific for epitope 4 showed an arginine residue in the N-terminal region of their TCRBV CDR3 loops despite their sequence diversities. In view of the striking heterogeneities found, therapeutical strategies aimed at the clonal deletion of allergen-specific T cell clones, providing help for IgE synthesis, may not be feasible. Moreover, 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↗
PubMed1996

The sequence and organization of the core histone H3 and H4 genes in the early branching amitochondriate protist Trichomonas vaginalis.

Among the unicellular protists, several of which are parasitic, some of the most divergent eukaryotic species are found. The evolutionary distances between protists are so large that even slowly evolving proteins like histones are strongly divergent. In this study we isolated cDNA and genomic histone H3 and H4 clones from Trichomonas vaginalis. Two histone H3 and three histone H4 genes were detected on three genomic clones with one complete H3 and two complete H4 sequences. H3 and H4 genes were divergently transcribed with very short intergenic regions of only 194 bp, which contained T. vaginalis-specific as well as histone-specific putative promoter elements. Southern blot analysis showed that there may be several more histone gene pairs. The two complete histone H4 genes were different on the nucleotide level but encoded the same amino acid sequence. Comparison of the amino acid sequences of the T. vaginalis H3 and H4 histones with sequences from animals, fungi, and plants as well as other protists revealed a significant divergence not only from the sequences in multicellular organisms but especially from the sequences in other protists like Entamoeba histolytica, Trypanosoma cruzi, and Leishmania infantum.

Amino Acid Sequence↗
PubMed1996

Food allergy to honey: pollen or bee products? Characterization of allergenic proteins in honey by means of immunoblotting.

OBJECTIVE: To characterize the allergenic components of honey, 23 patients allergic to honey were investigated. All displayed allergic symptoms after ingestion of honey or honey-containing products, ranging from itching in the oral mucosa to severe systemic symptoms to anaphylactic shock. METHODS AND RESULTS: Immunoblot analyses of the patients' sera revealed IgE binding to proteins at a molecular mass of 54 kd, 60 kd, 72 kd, or to a 30 kd/33 kd double band, or to both in sunflower honey extracts. The three bands corresponding to higher molecular mass proteins could also be detected in the three other kinds of honey (locust tree, European chestnut and forest honey) that were tested and represented bee products because IgE binding to these proteins was inhibited by extracts of honeybee heads and extracts of isolated bee venom sacs. The 30 kd/33 kd bands could be identified as sunflower honey-specific. When testing sera from patients allergic to bee venom with honey extracts, in seven of 10 cases IgE binding to bee-specific components could be observed. CONCLUSION: Both proteins derived from secretions of pharyngeal and salivary glands of honeybee heads and pollen proteins contained in the honey cause allergic reactions to honey.

Adolescent↗
PubMed1996

Induction of IgE antibodies with predefined specificity in rhesus monkeys with recombinant birch pollen allergens, Bet v 1 and Bet v 2.

BACKGROUND: Recombinant birch pollen allergens Bet v 1 and Bet v 2 (birch profilin) have been characterized in vitro previously. OBJECTIVE: To establish a close-to-man model of type I allergy, recombinant birch pollen allergens were injected into rhesus monkeys. METHODS: The allergens were expressed in Escherichia coli, purified to homogeneity and injected into rhesus monkeys with aluminium hydroxide as adjuvans. The development of type I allergy was monitored by measurement of specific IgE, in vitro histamine release tests, cellular proliferation assays, skin testing, and bronchial provocation tests. RESULTS: Immunized rhesus monkeys displayed symptoms of type I allergy comparable to those of allergic patients, and cross-reactivity of IgE antibodies with Bet v 1 and Bet v 2 homologous allergens was observed. Systemic application of corticosteroids during secondary immunizations suppressed specific antibody responses. CONCLUSION: Recombinant birch pollen allergens (Bet v 1 and Bet v 2) were effective to establish a close-to-man model of natural type I allergy in rhesus monkeys, allowing study of specific IgE regulation in vivo.

Allergens↗
PubMed1996

Sequence comparisons of the CDR3 hyper-variable loops of human T cell receptors specific for three major T cell epitopes of the birch pollen allergen Bet v 1.

We have analysed the T cell receptor (TCR) alpha and beta chain sequences of 16 human CD4+ T cell clones (TCCs) specific for three important epitopes of the major birch pollen allergen Bet v 1. The TCCs were raised from the peripheral blood of eight patients with birch pollen allergy, showing allergic rhino-conjunctivitis and allergic asthma. The TCCs from these individuals were specific for Bet v 1-derived peptides: amino acids (aa)77-92 (epitope 1), aa93-108 (epitope 2) and aa113-126 (epitope 3). The DNA sequence analysis of the TCRAV and BV regions revealed heterogeneous repertoires for recognition of the peptides. Multiple combinations of AV/AJ and BV/BJ were used. However, some inter-individual restriction was evident. A limited selection of AVS and the normally infrequently used BV1S4 was obvious in TCCs specific for epitope 1. The TCRBV13 was more frequent in TCCs recognizing epitope 3. A very narrow distribution in length could be seen in the CDR3 sequences of the beta chain of TCRs with specificity for epitopes 1 and 2. Inter-individual positional micro-restriction was observed for the aa motif LR in the tCDR3 (epitope 1), for the aa residue M in the alphaCDR3 and for the aa residue G in the betaCDR3 (epitope 3). Our results illustrate clearly that each antigenic peptide derived from a single allergen, is capable of selecting different characteristics in the responding repertoire of TCRs, thus increasing the complexity of allergen-recognition by T lymphocytes. Therefore, our findings limit the potential use of TCR targeted therapeutical strategies in Type I allergy.

Allergens↗
PubMed1996

Optimized growth medium for primary culture of human oral keratinocytes.

The effect of different media additives in defining optimal growth conditions for primary cultures of human oral keratinocytes was studied. A cocultivation technique with irradiated Swiss-3T3-fibroblasts in 96-well plates enables the comparison of additives for primary keratinocyte cultures derived from one patient. 3H-labeled thymidine uptake showed no growth or growth inhibition with adenine, choleratoxin or transferrin compared to basal medium (Dulbecco's modified Eagle's medium (DMEM) and 10% fetal calf serum). Among single additives, 5 micrograms/ml hydrocortisone, 5 micrograms/ml insulin, 10 ng/ml EGF, 2 micrograms/ml bovine pituitary extract, and 10(-9) M triiodothyronine showed the greatest capacity to promote keratinocyte growth. With all possible combinations of additives, maximum stimulation was found with a combination of EGF (10 ng/ml), insulin (5 micrograms/ml), and hydrocortisone (5 micrograms/ml); none of the other combinations were more effective. Our data indicate that in short-term cultures (up to 5 days) various media additives described in the literature are not necessarily required in this system of primary culture of human oral keratinocytes.

3T3 Cells↗
PubMed1996

Detection of allergen-specific IgE in tears of grass pollen-allergic patients with allergic rhinoconjunctivitis.

BACKGROUND: Allergic conjunctivitis is a common symptom amongst Type I (IgE-mediated) allergic diseases; and most frequently seen as rhinoconjunctivitis. However, the site of production and the significance of allergen specific IgE needs further elucidation. OBJECTIVE: We investigated whether the presence of IgE in tears of grass pollen allergic patients correlated with disease and clinical symptoms, whether the IgE binding pattern to the different grass pollen antigens was different in sera and tears, and whether IgA antibodies to grass pollen allergens were present in tears. Finally, we looked whether specific IgE was produced locally or was exudated from serum. METHODS: Sera from 44 grass pollen allergic patients suffering from either allergic rhinitis (n = 11) or allergic rhinoconjunctivitis (n = 33) and from healthy controls (n = 7) were used for the experiments. Binding of specific IgE and IgA antibodies to the different groups of grass pollen allergens (Phleum pratense) was evaluated by means of immunoblotting. RESULTS: Specific IgE was detected in sera as well as in tears of allergic patients, whereby tear-derived allergen-specific IgE exerted similar specificities to the corresponding IgE from serum. The correlation between symptoms of ocular allergy and the presence of allergen-specific IgE in tears was highly significant (P < 0.0001). In contrast, only a poor correlation was found between specific and/or total IgE in sera and the manifestation of ocular allergy (P = 0.73). CONCLUSION: Allergen-specific IgE antibodies in tears seem to be produced locally rather than exudated from serum. IgE in tears seems to be responsible for allergic conjunctivitis. IgA in tears cannot exert a protective function since the IgA antibodies recognize different antigens in a grass pollen (Phleum pratense) extract than IgE antibodies. The highly significant correlation between allergic conjunctivitis and the presence of specific tear IgE emphasizes the diagnostic value of immunoblots with tear IgE, especially in cases in which serum provides inconclusive results.

Allergens↗
PubMed1996

IgE cross-reactivity between birch pollen, mugwort pollen and celery is due to at least three distinct cross-reacting allergens: immunoblot investigation of the birch-mugwort-celery syndrome.

BACKGROUND: Allergy to celery is often associated with sensitization to birch and/or mugwort pollen. OBJECTIVE AND METHODS: In a multi-centre study, sera from 23 patients suffering from type 1 allergy to celery and 15 patients with positive celery RAST but no clinical sensitization were compared. To examine whether cross-reactivity between celery and mugwort pollen includes cross-sensitization to birch pollen allergens, we determined cross-reacting structures in birch pollen, mugwort pollen and celery by means of immunoblotting. Inhibition studies were performed by preincubation of sera with extracts of birch pollen, mugwort pollen, and celery. RESULTS: We identified three groups of proteins--homologues of Bet v 1 and birch profilin (Bet v 2) as well as a group of proteins with a molecular range of 46 to 60 kD--displaying IgE-cross-reactivity, which were shared by birch pollen and celery. Two of these groups of allergens (profilin and the 46 to 60 kD proteins) were also present in mugwort pollen. In this paper we demonstrate that most cross-reacting allergens present in mugwort pollen and celery can also be detected in birch pollen extract. CONCLUSION: Therefore we propose, from a serological point of view, to extend the mugwort-celery syndrome to the birch-mugwort-celery syndrome.

Allergens↗
PubMed1996

High-level expression of tree pollen isoallergens in Escherichia coli.

cDNAs coding for the major allergen of alder (Alnus glutinosa) pollen Aln g 1, for nine isoforms of Bet v 1, the major birch (Betula verrucosa) pollen allergen, and for four isoforms of Cor a 1, the major allergen of hazel (Corylus avellana) pollen, were inserted into the plasmid pMW175 or pMW 172 and expressed in Escherichia coli as recombinant non-fusion proteins. These constructs produced between 20 and 160 mg protein/l. The recombinant tree pollen isoallergens were tested in immunoblots for their antibody binding properties. For this purpose, we used two monoclonal antibodies (BIP 1 and BIP 4) raised against natural Bet v 1, a polyclonal rabbit anti-recombinant Bet v 1a, as well as serum IgE from allergic patients. Our results show that this expression system is suitable for the production of milligram amounts of tree pollen isoallergens which can be used for the characterization of allergenic epitopes recognized by T and B cells.

Allergens↗
PubMed1996

2-D protein crystals as an immobilization matrix for producing reaction zones in dipstick-style immunoassays.

In the present study, the applicability of crystalline bacterial cell-surface layers (S-layers) as novel immobilization matrices and reaction zones for dipstick-style immunoassays was investigated. For this purpose, S-layer-carrying cell-wall fragments from Bacillus sphaericus CCM 2120 were deposited on a microporous support, and the S-layer protein was cross-linked with glutaraldehyde. For developing appropriate test systems, either human IgG was directly linked to the carboxylic acid groups from the S-layer protein or it was immobilized using Protein A or, after biotinylation, using streptavidin. A clear correlation was obtained between the amount of anti-human IgG applied and the absorbance values in the immunoassays. S-layers with covalently bound recombinant major birch pollen allergen were used for quantitative and semiquantitative determination of an antibody raised against it. Using S-layers as an immobilization matrix in comparison to amorphous polymers has advantages in that the closed monolayers of functional macromolecules on their outermost surface allows for strong signals in immunoassays, almost completely eliminates background and prevents diffusion.

Antibodies, Monoclonal↗