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D Kraft

Publications and source records attributed to D Kraft.

At least 109 records · Page 6Linked to original sources
PubMed1997

Recombinant allergen-specific antibody fragments: tools for diagnosis, prevention and therapy of type I allergy.

Type I allergy represents a hypersensitivity occurring in almost 20% of the population that is based on the recognition of innocuous airborn antigens (pollen, mite, mould and pet allergens) by specific immunoglobulin E. Allergic symptoms (e.g. allergic rhinitis, conjunctivitis, asthma) are caused by the release of biological mediators from effector-cells after allergen-induced crosslink of receptor-bound IgE. Here we discuss strategies to obtain recombinant allergen-specific antibody fragments (Fabs) from mouse and human cell lines as well as directly from allergic patients lymphocytes via the combinatorial library technology. It is suggested to use recombinant allergen-specific Fabs for the standardization of allergen extracts currently used for diagnosis and treatment, to determine allergen contents in allergen sources and the environment to allow preventive measures and to use allergen-specific Fabs as therapeutic tools to interfere with the allergen-IgE interaction. The latter appears possible because IgE represents the least abundant class of immunoglobulins and there is increasing evidence for a limited diversity among allergens and their B-cell epitopes. Moreover, allergic effector reactions are mostly confined to accessible target organs so that a local application of competing Fabs prior to allergen exposure might represent a feasible therapeutic approach.

Allergens↗
PubMed1997

Cloning allergen-specific antibody fragments (Fabs); tools for allergen standardization and therapy of type I allergy.

Recombinant allergens have made it possible to dissect the mechanisms of allergen-antibody interactions at a molecular level. It becomes clear that monoclonal human IgG antibodies as well as animal derived antibodies can block the interaction of specific IgE antibodies as well as the allergen induced allergic effector reaction. Using PCR technology and combinatorial plasmid vectors, recombinant antibody fragments can be produced and it has even become possible to isolate allergen-specific IgE Fabs out of combinatorial IgE libraries constructed from allergic patients lymphocytes. Recombinant Fabs will represent useful tools to study the IgE-allergen interaction as well as for the standardization of allergen extracts and quantitative allergen measurements. Moreover, allergen-specific recombinant Fabs which block the allergen-IgE interaction have to be considered as tools for local therapy in effector organs of allergic patients.

Allergens↗
PubMed1997

Characterization of allergens in Apiaceae spices: anise, fennel, coriander and cumin.

BACKGROUND: Symptoms elicited by IgE-mediated food allergy range from mild local to severe systemic reactions. Allergens in spices are particularly dangerous due to their hidden presence in many dishes. OBJECTIVES AND METHODS: According to clinical observations, mugwort and birch pollen allergy, and hypersensitivity to spices are frequently associated, but the crossreacting compounds were not defined so far. We tested sera of 15 patients who experienced adverse reactions to spiced food and characterized their IgE-binding patterns on anise, fennel, coriander and cumin extracts through immunoblot and inhibition experiments. RESULTS: The use of anti-Bet v 1 (MoAb) and anti-profilin (rabbit) antibodies revealed the presence of crossreacting allergens in the tested spice extracts. Inhibition experiments showed that IgE-binding to allergens in Apiaceae spices could be blocked by preincubation of sera with rBet v 1 or rBet v 2 (birch profilin). Moreover, we detected crossreacting allergenic molecules in the molecular weight range of 60 kDa. IgE-binding to spice allergens occurred only with sera of 10/15 (66%) patients with allergy to pollen (birch, mugwort) and/or celeriac. In five out of 15 (33%) patients with a history of adverse reaction to spices, but without pollen and celeriac allergy, no IgE-binding to any spice protein could be demonstrated. It is possible that these clinical reactions could be elicited by other types of hypersensitivity (Type II, III, IV), however, as spices contain highly reactive substances, the symptoms may most likely be classified as food-intolerant. CONCLUSIONS: Bet v 1- and profilin-related allergens may, besides higher molecular weight allergenic molecules, be responsible for Type I allergy to anise, fennel, coriander or cumin, members of the Apiaceae.

Adult↗
PubMed1996

Molecular characterization of Bip 1, a monoclonal antibody that modulates IgE binding to birch pollen allergen, Bet v 1.

Bet v 1 and homologous proteins represent major cross-reactive allergens for more than 95% of tree pollen-, fruit-, and vegetable-allergic individuals. To study the interaction of Bet v 1 and the immune system, we characterized a Bet v 1-specific mAb, Bip 1. Soluble rBip 1 Fabs were expressed in Escherichia coli and purified by affinity chromatography using immobilized Bet v 1. Bip 1 Fabs displayed a cross-reactivity to homologous allergens comparable with that of IgE Abs from allergic patients. Preincubation of Bet v 1 with Bip 1 led to an up to fivefold increase of allergic patients' IgE binding to Bet v 1. This enhancement in IgE binding may be interpreted as stabilization of a Bet v 1 state, in which certain IgE epitopes are better applicable. It also shows that allergic patients possess IgE Abs directed against different Bet v 1 conformations. The modulation of Ab binding to a given Ag by other Abs was observed also for human Bet v 1-specific IgG Abs, and may represent a novel mechanism for the regulation of specific humoral immune responses in a complex network.

Allergens↗
PubMed1996

Molecular and structural analysis of a continuous birch profilin epitope defined by a monoclonal antibody.

The interaction of a mouse monoclonal antibody (4A6) and birch profilin, a structurally well conserved actin- and phosphoinositide-binding protein and cross-reactive allergen, was characterized. In contrast to serum IgE from allergic patients, which shows cross-reactivity with most plants, monoclonal antibody 4A6 selectively reacted with tree pollen profilins. Using synthetic overlapping peptides, a continuous hexapeptide epitope was identified. The exchange of a single amino acid (Gln-47 --> Glu) within the epitope was found to abolish the binding of monoclonal antibody 4A6 to other plant profilins. The NMR analyses of the birch and the nonreactive timothy grass profilin peptides showed that the loss of binding was not due to major structural differences. Both peptides adopted extended conformations similar to that observed for the epitope in the x-ray crystal structure of the native birch profilin. Binding studies with peptides and birch profilin mutants generated by in vitro mutagenesis demonstrated that the change of Gln-47 to acidic amino acids (e.g. Glu or Asp) led to electrostatic repulsion of monoclonal antibody 4A6. In conclusion the molecular and structural analyses of the interaction of a monoclonal antibody with a continuous peptide epitope, recognized in a conformation similar to that displayed on the native protein, are presented.

Allergens↗
PubMed1996

High-level expression in Escherichia coli and purification of recombinant plant profilins: comparison of IgE-binding capacity and allergenic activity.

Because of their structural similarity and ubiquitous distribution as actin binding proteins, plant profilins represent important cross-reactive allergens for almost 20% of patients suffering from Type I allergy to pollen and other plant products. The cDNAs coding for three birch profilin variants (Tyr44, Glu47, and Asn47), timothy grass profilin, and three tobacco profilin isoforms (ntprof1-3) were expressed at high levels in Escherichia coli as non-fusion proteins. The recombinant plant profilins were purified to homogeneity by poly (L-proline) affinity chromatography and showed comparable capacity to bind IgE-antibodies from profilin allergic patients. All recombinant plant profilins elicited dose-dependent histamine release from basophils of a profilin allergic patient and induced immediate type skin reactions. It is concluded that profilins from different plant species share IgE-epitopes and allergenic properties. Plant profilins therefore constitute a family of functional pan-allergens which may substitute each other for diagnosis and specific immunotherapy.

Allergens↗
PubMed1996

Secondary structure and tertiary fold of the birch pollen allergen Bet v 1 in solution.

Bet v 1 is the major birch pollen allergen and therefore the main cause of type I allergies observed in early spring. It is composed of 159 amino acid residues adding up to a molecular mass of 17 kDa. We determined the secondary structure and tertiary fold of full-length Bet v 1 by NMR spectroscopy. Two- and three-dimensional NMR measurements suggest that Bet v 1 is a globular monomer in solution with a high content of well defined secondary structure. Of the total of 159 residues, 135 could be sequentially assigned, using an improved assignment strategy based mainly on heteronuclear experiments. An improved strategy for structure calculation revealed three helices and two beta-sheets as major elements of secondary structure. The globular tertiary structure is mainly stabilized by two antiparallel beta-sheets. The two helices at the C terminus are in accordance with the results from the solution structure of the chemically synthesized peptide Bet v 1-(125-154). This peptide is composed of two helices connected by a hinge. The structural features of Bet v 1 are highly similar to those found in the Ambrosia allergen Amb t V.

Allergens↗
PubMed1996

Peripheral blood dendritic cells express Fc epsilon RI as a complex composed of Fc epsilon RI alpha- and Fc epsilon RI gamma-chains and can use this receptor for IgE-mediated allergen presentation.

Originally limited to basophils and mast cells, the spectrum of high affinity IgE receptor (Fc epsilon RI-bearing cells has expanded recently to include Langerhans cells, dermal dendritic cells (DC), monocytes, and eosinophils. As a result of studies on the distribution, structure, and function of Fc epsilon RI on APCs, we discovered a minor nonbasophil, nonmonocyte PBMC population that can bind IgE via Fc epsilon RI. This receptor occurs on the surface of these cells as a multimeric structure containing Fc epsilon RI alpha- and Fc epsilon RI gamma-chains but, unlike its counterpart on basophils, lacking Fc epsilon RI beta. Further experiments revealed that these Fc epsilon RI alpha gamma-expressing cells closely resemble peripheral blood DC by immunophenotype (HLA-DRhigh, HLA-DQhhigh; CD4+, CD11a+, CD32+, CD33+, B7/2 (CD86)+; CD11blow, CD14low, CD40low, CD54low, CD64low) and cell morphology. These features allowed us to isolate Fc epsilon RI-expressing DC from the peripheral blood and to investigate their immunostimulatory properties. We found Fc epsilon RI-positive DC to be efficient stimulators of both primary (allogeneic MLR) and Fc epsilon RI/IgE-dependent, secondary T cell responses at low cell numbers. Thus, Fc epsilon RI-expressing DC may not only amplify established type I allergic immune reactions but, unlike Fc epsilon RI-positive semiprofessional APCs, may be able to prime naive T cells to common and/or cryptic epitopes of IgE-reactive Ags.

Allergens↗
PubMed1996

Human IgG monoclonal antibodies that modulate the binding of specific IgE to birch pollen Bet v 1.

Birch pollen allergy is a very frequent pathology in Europe and North America. More than 95% of the tree pollen allergic patients display IgE reactivity against Bet v 1, the major birch pollen allergen. Starting with PBL from a patient desensitized by immunotherapy, we have generated five B cell lines (BAB1 to BAB5) that secrete human IgG mAbs of high affinity for Bet v 1. Although competition studies indicated that these human IgG mAb recognized different epitopes, broad cross-reactivity was found with Bet v 1 homologous allergens present in tree pollens and plant-derived foods. When tested for interference with allergic patients' IgE, BAB1 inhibited (by 80-100%) the binding of IgE to nitrocellulose-blotted Bet v 1, while BAB2 enhanced it. The biologic significance of the ability of BAB1 to interfere with patients' IgE binding is indicated by the finding that BAB1 completely inhibited Bet v 1-induced histamine release from allergic patients' basophils. Allergen-specific human IgG mAbs such as BAB1, which presents high blocking activity in both immunochemical and cellular IgE competition experiments, might have therapeutical application.

Adjuvants, Immunologic↗
PubMed1996

Evidence for an alpha helical T cell epitope in the C-terminus of the main birch pollen allergen Bet V 1.

Secondary structure prediction of the main birch pollen allergen Bet v 1 was found to be in good agreement with the secondary structural elements found by analysing the Bet v 1 circular dichroism data. According to both experiment and prediction, 32% of 160 amino acids participate in alpha helices, 21% in beta sheets, 24% in turns, and 23% in other structural motifs. The peptide LRAVESYLLAHS which represents one of the major T cell epitopes on Bet v 1 was shown to have a high propensity to form an alpha helix. Time-resolved fluorescence anisotropy measurements of the allergen revealed an overall rotational correlation time of 7.35 ns, which corresponds to a hydrodynamic molecular radius of 19.2 A. This refers to a monomeric Bet v 1 molecule in solution, which is also reflected in the narrow band width of the 1H-NMR spectrum. The results presented here are in good agreement with the recently solved NMR structure of Amb t 5: both allergens are monomers in solution with an extended C-terminal alpha helix containing a major T cell epitope.

Allergens↗
PubMed1996

Construction of a combinatorial IgE library from an allergic patient. Isolation and characterization of human IgE Fabs with specificity for the major timothy grass pollen allergen, Phl p 5.

To characterize human IgE antibodies with specificity for a major allergen at the molecular level, we have constructed an IgE combinatorial library from a grass pollen allergic patient. cDNAs coding for IgE heavy chain fragments and for light chains were reverse-transcribed and polymerase chain reaction-amplified from RNA of peripheral blood lymphocytes and randomly combined in plasmid pComb3H to yield a combinatorial library of 5 x 10(7) primary clones. IgE Fabs with specificity for Phl p 5, a major timothy grass pollen allergen, were isolated by panning. Sequence analysis showed that the 4 of the Fabs used the same heavy chain fragments which had combined with different kappa light chains. Soluble recombinant IgE Fabs were purified by affinity chromatography to Phl p 5 and, like natural IgE antibodies, cross-reacted with group 5 allergens from different grass species. The described approach should facilitate studies on the molecular interaction between IgE antibodies and allergens and encourages the consideration of specific IgE Fabs that are capable of interfering with allergen-IgE binding as potential therapeutic tools.

Allergens↗
PubMed1996

Expression of Zm13, a pollen specific maize protein, in Escherichia coli reveals IgE-binding capacity and allergenic potential.

Plant proteins belong to the most frequent elicitors of type I allergic symptoms in industrialized countries. Several relevant plant allergens have been found to be either specifically expressed or highly upregulated in mature pollen. The cDNA coding for a pollen specific maize protein, Zm13, shows significant sequence homology with a number of pollen or anther specific proteins from monocot and dicot plants as well as with recently described allergens from olive and rye grass. To test whether the Zm13 protein might possess IgE-binding capacity, Zm13 was expressed in E. coli. The coding region of Zm13 was PCR amplified from a genomic clone and expressed as a glutathione-S-transferase fusion protein. The recombinant Zm13 fusion protein bound a Zm13 specific rabbit antiserum and reacted with serum IgE from grass pollen allergic patients indicating that Zm13 and homologous proteins represent a family of conserved plant allergens.

Allergens↗
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↗