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Biomedical subjects

O Scheiner

Publications and source records attributed to O Scheiner.

216 records · Page 12Linked to original sources
PubMed1979

Purine metabolism in human lymphocytes.

In peripheral human blood lymphocytes the uptake and metabolism of adenine, guanine, and hypoxanthine was investigated. This was achieved by incubation of purified lymphocytes with 14C-purine bases, separation of cells from the incubation medium by a rapid filtration technique, and subsequent separation of the acid soluble material by thin-layer chromatography. No perferential uptake for one of the purine bases was observed. In all cases only traces of 14C-purine bases not added originally and labeled nucleosides could be demonstrated. Approximately 2/3 of adenine and 1/2 of guanine or hypoxanthine were converted to nucleotides. Separation of formed nucleotides showed that adenine and guanine were metabolized mainly to their corresponding nucleotides; hypoxanthine was converted to a considerable amount to adenine nucleotides and only to a small proportion into its own nucleotides. These results demonstrate the predomonance of adenine nucleotide formation in normal human lymphocytes.

Adenine↗
PubMed1978

Separation and characterization of anti-benzylpenicilloyl (BPO) antibodies. I. Biochemical and biophysical properties of anti-BPO-IgG obtained by affinity and subsequent ion-exchange chromatography.

Anti-BPO antibodies were purified by means of affinity chromatography using AH-Sepharose 4B coated with covalently bound BPO groups. Specific elution was achieved by the hapten analogue BPO-epsilon-aminocaproic acid (BPO-EACA); desorption of the remaining antibody was performed thereafter by 0.1 M acetic acid. The resulting antibody fractions--hapten-eluted antibody (H-Ab) and acid eluted antibody (A-Ab), respectively--were further separated by ion-exchange chromatography which led to the appearance of 3 subfractions in the case of H-Ab (H1, H2, H3) and 2 subfractions in the case of A-Ab (A1 and A2). In liquid isoelectrofocusing an inhomogeneous pattern resulted. The bulk of antibodies focused between pH 6.5 and 7.0. The average avidity of H-Ab was found to be higher than that of A-Ab suggesting that avidity may influence the elution pattern in affinity chromatography. The hydrophobic influence of the "spacer" and/or interactions of antibodies directed against the hydrophobic regions of the BPO group may explain why a considerable part of the antibodies could be recovered from the immunosorbent only by acid elution.

Animals↗
PubMed1978

Separation and characterization of anti-benzylpenicilloyl (BPO) antibodies. II. Immunological properties of different IgG fractions.

Affinity chromatography and subsequent ion-exchange chromatography of pooled anti-benzylpenicilloyl (anti-BPO) hyperimmune sera separated 5 different anti-BPO IgG fractions as described in the preceding paper. These fractions were tested for activities in passive hemagglutination (PHA), passive immune hemolysis (PIH), antibody-dependent cellular cytotoxicity (ADCC) and influence on IgM-induced hemolysis. It was found that anti-BPO IgG fractions with low avidity (dissociation constant K = 4.4--6.7 X 10(-8) moles/l) were poorly active in PHA and ADCC and had no blocking activity in IgM hemolysis. Among the highly avid antibodies (K = 0.7--3.4 X 10(-8) mole/l) no correlations were found between avidity and activities in the immunological tests. The results presented demonstrate that ion-exchange chromatography allows the separation of blocking and lytic antibodies as shown by their influence on IgM-induced complement-dependent lysis of lightly hapten-coated sheep erythrocytes.

Analysis of Variance↗
PubMedDate not supplied

Antibody-dependent killer-cell function in insulin-dependent diabetes mellitus.

Antibody-dependent killer-cell activity (ADCC) was determined in 36 insulin-dependent diabetics (IDD) and 32 controls. The medians of cytotoxic indices obtained by using either antibody coated chicken red blood or HeLa cells as targets were statistically significantly reduced in the diabetics (P less than 0.05 for both systems). However, due to the wide range and considerable overlap of the cytotoxic indices observed in patients and controls, the biological significance of these mathematical differences remains to be determined. The duration of the disease did not have any influence on killer (K) cell function and only a slight tendency for decreased ADCC during episodes of poor metabolic control was noted. Further analysis of the influence of diabetes-associated factors did not reveal any definite correlation between the functional K cell deficit, the insulin dosage administered, the insulin antibody titers, the blood glucose at the time of sampling, the 24 hour glucosuria and IDD-associated immunogenetic factors.

Adult↗
PubMedDate not supplied

Molecular characterization of the cDNA coding for translation elongation factor-2 of pathogenic Entamoeba histolytica.

To investigate the humoral immune response of patients with amoebic dysentry against Entamoeba histolytica, immunoglobulin G (IgG)-immunopositive cDNA clones from the pathogenic strain SFL-3 were examined. A large part of the IgG-positive cDNA clones obtained with one serum encoded highly conserved intracellular proteins. A clone was found that was homologous to translation elongation factor-2 (EF-2). Sequence analysis of the EF-2 cDNA showed 63.6% amino acid sequence identity with the human homologue. The deduced protein sequence has a length of 840 amino acid residues with a molecular mass of 93.3 kD. The 3' and 5' untranslated regions of the mRNA are relatively short as shown for other genes of E. histolytica. A genomic clone was used to analyze the region upstream of the translation initiation codon. The codon distribution of EF-2 and other published E. histolytica sequences reflects the high A/T content. The codons for different amino acids are biased to a widely differing extent.

Amino Acid Sequence↗
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↗
PubMedDate not supplied

Characterization of allergen (Bet v 1)-specific T cell lines and clones from non-allergic individuals.

The immune response towards allergens in non-allergic healthy individuals was investigated. T cell lines (TCL) with specificity for Bet v 1, the major birch pollen allergen, were established and analysed for epitope specificity. 49 T cell clones (TCC) specific for Bet v 1 were isolated from TCLs. All TCCs revealed the Th phenotype. Cytokine production in response to specific stimulation revealed a majority of Th clones producing interleukin (IL)-4 and interferon (IFN)-gamma; however, most TCCs revealed a low IL-4/IFN-gamma ratio. Immunoblot revealed Bet v 1-specific IgG in non-allergic individuals whereas no IgE could be detected. Our results indicate that T cells from allergic and non-allergic individuals recognize the same epitopes on allergenic molecules, leading to activation, which then results in a differential production of cytokines and consequently to differential isotype switching in allergen-specific B cells.

Allergens↗
PubMedDate not supplied

Cross-reacting allergens in tree pollen and pollen-related food allergy: implications for diagnosis of specific IgE.

BACKGROUND: A number of recombinant allergens are by now constituents of devices that can be routinely used for the detection of specific IgE. Therefore, the results of diagnostic procedures using conventional allergen extracts can be compared with those employing selected recombinant allergens. METHODS: Thirty-four sera from patients allergic to birch pollen were tested with the standard t3-CAP and rBet v 1a- and rBet v 2-CAP. cDNA was prepared by RT-PCR using primers according to the N terminus of purified allergens. Expression cDNA libraries were screened with IgE from selected patients. RESULTS: Twenty-four patients allergic to birch pollen showed the same RAST class with t3 as with rBet v 1a; 8 patients differed within 1 RAST class. In addition, 3 patients showed RAST class 3 with rBet v 2. Besides Bet v 1 and Bet v 2, 3 allergens from celery and avocado belonging to highly conserved protein families were cloned and sequenced. CONCLUSIONS: rBet v 1a can be expected to represent an excellent tool for the diagnosis of patients allergic to birch pollen in Central, Northern, and Eastern Europe. Still, a much higher number of patients has to be tested. For their high degree of conservation, further protein families have to be identified to explain cross-reactivities of birch pollen allergens other than Bet v 1 and Bet v 2 with, e.g., allergens from vegetable food.

Allergens↗
PubMedDate not supplied

Isoforms of atopic allergens with reduced allergenicity but conserved T cell antigenicity: possible use for specific immunotherapy.

BACKGROUND: We analyzed the T cell activation potency and the IgE-binding properties (allergenicity) of nine isoforms of Bet v 1, the major allergen of birch pollen. METHODS: The capacity of recombinant Bet v 1 isoforms to bind serum IgE from allergic patients was evaluated by immunoblot experiments and skin prick tests. The potency of Bet v 1 isoallergens to activate T lymphocytes from birch-pollen-allergic patients was assayed using allergen-specific T cell clones. RESULTS: According to their ability to bind IgE from allergic patients in immunoblot experiments, Bet v 1 isoforms can be grouped into high-IgE-binding molecules and molecules with low/no IgE-binding activity. Representatively, isoform d was used in skin tests. Skin prick tests revealed no potency of this isoform to induce wheal and flare reactions in the skin of birch-pollen-allergic individuals. In contrast, isoform a and natural Bet v 1 displayed high allergenicity in vivo. On the other hand, Bet v 1 isoform d (low allergenicity) displayed significant higher T cell activation potency when compared to isoform a (high allergenicity). CONCLUSION: Based on these findings, we propose a new form of specific immunotherapy using hypoallergenic recombinant allergen isoforms.

Allergens↗
PubMedDate not supplied

T cell receptor CDR3 sequences and recombinant T cell receptors. Prerequisites for developing ligands to interfere with T cell receptor binding to the MHC/peptide complex.

BACKGROUND: The interaction of T cell receptors (TCRs) with peptide fragments bound to major histocompatibility complex (MHC) molecules is central to the initiation and propagation of most immune responses. In order to understand and control the molecular interactions underlying T cell recognition of MHC/peptide complexes, recent efforts have focused on the production of recombinant soluble forms of the TCR heterodimer. METHODS: TCRA variable (TCRAV) and TCRBV sequences used by human T cell clones were amplified by PCR, cloned and sequenced. The deduced amino acid sequences of the complementarity determining region (CDR) 3 loops of TCRs specific for the same allergenic epitope were compared. V region genes of a selected TCR were expressed as a single-chain (sc) molecule in the periplasm of Escherichia coli. RESULTS: Conserved amino acid motifs specific for allergenic peptides of Bet v 1 and Phl p 1 were identified in CDR3 sequences of TCRs. A recombinant scTCR was produced. The ratio of insoluble to soluble material was 1:1. The recombinant protein was of the correct size and showed no signs of degradation. CONCLUSIONS: Conservation of amino acid motifs in CDR3 loops of TCRs specific for the same allergen fragments indicated that the three-dimensional structure of the CDR3 was determined by the presented peptide. The recombinant scTCR will be used to identify ligands for the CDR3s from random peptide libraries to interfere with TCR binding to the MHC/peptide complex.

Allergens↗