Biomedical subjects

D Kraft

Publications and source records attributed to D Kraft.

At least 19 recordsLinked to original sources

PubMed1997

Conversion of the major birch pollen allergen, Bet v 1, into two nonanaphylactic T cell epitope-containing fragments: candidates for a novel form of specific immunotherapy.

A novel approach to reduce the anaphylactic activity of allergens is suggested. The strategy makes use of the presence of conformational immunoglobulin E (IgE) epitopes on one of the most common allergens. The three dimensional structure of the major birch pollen allergen, Bet v 1, was disrupted by expressing two parts of the Bet v 1 cDNA representing amino acids 1-74 and 75-160 in Escherichia coli. In contrast to the complete recombinant Bet v 1, the fragments showed almost no allergenicity and exhibited random coil conformation as analyzed by circular dichroism. Both nonanaphylactic fragments induced proliferation of human Bet v 1-specific T cell clones, indicating that they harbored all dominant T cell epitopes and therefore may be considered as a basis for the development of a safe and specific T cell immunotherapy.

Allergens↗

PubMed1997

High-level expression and purification of the major birch pollen allergen, Bet v 1.

Bet v 1, the single major allergen from birch pollen, shares IgE epitopes with all major tree pollen allergens from closely related species such as alder, hazel, hornbeam, beech, and European chestnut. Because of high sequence homologies among these allergens and the well-studied cross-reactivities on B cell epitopes, Bet v 1 is a representative model protein which can be used for in vitro studies. The cDNA coding for Bet v 1, the single major allergen from birch pollen, was cloned into the T7-based Escherichia coli expression system pMW 175/BL21(DE3) and synthesized as a nonfusion protein. In contrast to other E. coli systems (e.g., pKK233-2/JM105), this system produces high levels of readily extractable proteins corresponding to 5-10% of E. coli total protein, the percentage varying with culture conditions. The overall yield was 8-10 mg of purified recombinant protein per liter of culture medium. The recombinant allergen was purified by several steps, including ion-exchange and hydrophobic interaction chromatography. The purified recombinant allergen showed identical immunological properties with the respective natural counterpart. The use of recombinant allergens of high purity is expected to result in more accurate diagnostic procedures, but possibly also in a superior immunotherapy of Type I allergic diseases when compared with methods using crude allergen extracts containing various amounts of allergen concentrations.

Allergens↗

PubMed1997

Recombinant Bet v 1, the major birch pollen allergen, induces hypersensitivity reactions equal to those induced by natural Bet v 1 in the airways of patients allergic to tree pollen.

BACKGROUND: Atopic allergens produced by recombinant DNA methods are promising tools for diagnosis and therapy of Type I allergy. To evaluate the immunologic properties of these molecules, it is necessary to compare them with natural allergens in vitro and in vivo. OBJECTIVE: The study was carried out to determine whether the potency of recombinant Bet v 1 (rBet v 1) is comparable to that of natural Bet v 1 (nBet v 1) in inducing allergic reactions in the nose and bronchi. METHODS: Thirteen patients allergic to birch pollen with bronchial asthma and/or rhinitis were investigated. Skin prick tests and nasal and bronchial challenges were performed with rBet v 1 and nBet v 1. RESULTS: In patients allergic to birch pollen, both allergens induced comparable skin reactions. In subjects with rhinitis rBet v 1 was equally potent in inducing nasal reactions (mean PD(+60)NR +/- SD, 10.48 +/- 17.42 microg vs 7.98 +/- 8.9 microg, p > 0.05). In patients with asthma, rBet v 1 was equally potent in inducing bronchial reactions (PD20 FEV1, 0.81 +/- 1.74 microg vs 0.62 +/- 1.44 microg, p > 0.05) as nBet v 1. CONCLUSION: No significant differences were observed between natural and recombinant allergen. We conclude that allergens produced by recombinant techniques can induce typical allergic reactions in important target organs of Type I allergy: the nose and bronchi.

Adult↗

PubMed1997

Major cat and dog allergens share IgE epitopes.

BACKGROUND: Patients allergic to cats and dogs frequently display IgE reactivity against allergens from different animals, suggesting a cross-sensitization to common allergenic determinants. Although albumins have been recognized as relevant cross-reactive allergens, little is known regarding cross-reactive epitopes of the major cat and dog allergens. OBJECTIVE: In this study, sera from patients allergic to cats and/or dogs were used to investigate the presence of common IgE epitopes among the major cat and dog allergens. METHODS: The IgE reactivity profile of 109 patients who were allergic to allergens from several species of animals was determined with nitrocellulose-blotted cat and dog allergens. Sera from patients who were strongly allergic to the major cat and dog allergens were tested for the presence of cross-reactive IgE antibodies by one-dimensional and two-dimensional immunoblot inhibition experiments and by quantitative measurements obtained with the CAP-FEIA system (Pharmacia). RESULTS: Sixty-eight of 109 patients with animal allergy showed IgE reactivity to cat allergens and dog allergens. Sera from patients with both cat and dog allergy detected allergens of similar molecular weight in nitrocellulose-blotted cat and dog hair/dander extracts. Common, as well as species-restricted, IgE epitopes of the major cat and dog allergens could be demonstrated by IgE inhibition studies. CONCLUSION: Shared IgE epitopes of the major cat and dog allergens may provide an explanation for the clinical observation that allergies to cats and dogs are frequently associated.

Albumins↗

PubMed1997

Modulation of the allergic immune response in BALB/c mice by subcutaneous injection of high doses of the dominant T cell epitope from the major birch pollen allergen Bet v 1.

Several in vitro and in vivo studies indicate that application of high doses of dominant T cell epitopes can induce a state of antigen-specific non-responsiveness (anergy). In the present study, we developed a murine model of an allergic immune response to Bet v 1, the major birch pollen allergen. Mice were sensitized by injection of rBet v 1 and the allergic state was proven by the presence of allergen-specific IgE and positive immediate-type skin tests to Bet v 1. In epitope mapping experiments, an immunodominant T cell epitope of Bet v 1 in BALB/c mice was identified by the use of overlapping peptides. This peptide (BV 139) was subsequently employed for treatment. Two tolerization protocols were used: in one approach, the peptide was administered to naive mice before immunization (group BV139-S), in the second, already sensitized mice were treated (S-BV139). The results demonstrated that administering high doses of the dominant T cell epitope of Bet v 1 profoundly diminished T cell proliferation to the peptide in the BV139-S group, and to the peptide as well as to the whole protein in the S-BV139 group. Skin test reactivity to Bet v 1 was reduced in the BV139-S group. However, no differences in terms of specific antibody production between treated and untreated mice could be observed. This study provides evidence that administration of dominant T cell epitopes can down-regulate the allergen-specific T cell response. Proceeding on the assumption that the T lymphocyte response to allergens is crucial for the induction and maintenance of the allergic disease, a modulation of the immune response to allergens by treatment with T cell epitope peptides could represent a promising concept for immunotherapy in the future.

Adjuvants, Immunologic↗

PubMed1997

Food allergy to pumpkinseed--characterization of allergens.

In recent years, pumpkinseed has become increasingly popular as a foodstuff. Here we report the occurrence of allergic reactions (itching and swelling of oral mucosa, and asthma) to this member of the Cucurbitaceae family. We investigated three patients suffering from symptoms after ingestion of roasted pumpkinseed. All the patients fished for sport and used pressed pumpkinseed flour as bait. Sera were tested by the immunoblot technique for IgE reactivity with proteins of pumpkinseed extract. The immunoblot revealed pumpkinseed allergens of 13, 14, 36, 48, 77, and 87 kDa. Inhibition experiments with recombinant birch profilin were performed: IgE binding to the 14-kDa allergen was completely blocked by preincubation of the sera with recombinant birch profilin. In conclusion, type I allergy to pumpkinseed is rare, and the patients' histories suggest inhalation of pumpkinseed flour during fishing to be the relevant route of sensitization, leading to food allergy to pumpkinseed.

Adult↗

PubMed1997

Persistent pruritus after hydroxyethyl starch infusion therapy: a result of long-term storage in cutaneous nerves.

A high incidence of severe pruritus has been observed after the administration of hydroxyethyl starch (HES) on account of plasma volume substitution and improvement of the microcirculation. The aim of this study was to elucidate the possible pathomechanisms of HES-induced itching. Skin biopsies were taken from 93 patients, half of them presenting with pruritus, who received HES of various preparations and cumulative dosages. The samples were subjected to immunoelectron microscopical investigation using an antibody highly specific for HES. After infusion therapy with HES, formation of intracytoplasmic storage vacuoles in the skin could be demonstrated in all patients. A dose-dependent uptake of HES was first detectable in macrophages and, thereafter, in endothelial and epithelial cells. Consecutive control biopsies taken from single patients revealed a subsequent reduction of the vacuoles, in size and number, within 3 years, thus indicating a regular cutaneous metabolism of HES. Patients suffering from pruritus consistently showed additional deposition of HES in small peripheral nerves. HES-reactive vacuoles could be demonstrated in the Schwann cells of unmyelinated, as well as small myelinated, nerve fibres, and in endoneural and perineural cells. Neural devacuolization paralleled the clinical improvement in the symptoms. In conclusion, HES deposits in cutaneous nerves, as a consequence of a higher cumulative dosage, may account for the itching seen after HES infusion.

Adolescent↗

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↗