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B M Stadler

Publications and source records attributed to B M Stadler.

At least 37 records · Page 2Linked to original sources
PubMed1998

Epitope-specific antibody response to IgE by mimotope immunization.

We have previously described a mouse monoclonal anti-human IgE antibody (BSW17) capable of recognizing receptor-bound IgE without inducing mediator release from human basophils or mast cells. Moreover, immune complexes of IgE and BSW17 are not able to bind to the IgE receptor. An initial attempt to map the precise epitope recognized by this mAb by using Fc epsilon-derived peptides of variable length was unsuccessful. However, by screening random peptide phage display libraries we isolated circular nona- and octapeptides specifically recognized by BSW17. These constrained peptides mimic at least a part of a conformational epitope and are thus called mimotopes. These mimotopes, either phage displayed or synthetically synthesized, did not react with any other anti-human IgE antibody tested, but efficiently inhibited the binding of human IgE to BSW17 only. The use of Rhodol-Green-labeled free cyclic peptide proved that these interactions were not carrier dependent. Immunization of rabbits with phage clones displaying the specific peptides on the surface induced an anti-human IgE response specific for the epitope of BSW17. Therefore, we conclude that such mimotopes or mimotope-derived peptides might be used for vaccination to induce in vivo a beneficial anti-IgE response as a novel immunotherapy.

Amino Acid Sequence↗
PubMed1998

A mimotope defined by phage display inhibits IgE binding to the plant panallergen profilin.

Birch pollen and mugwort pollen allergies are often associated with hypersensitivity to plant foods. This clinical and serological cross-reactivity is mediated by IgE antibodies reacting with homologous proteins in pollen and food. Cross-reacting homologs of the important birch pollen allergen Bet v 2 (profilin) could be detected in other pollen, fruits, nuts, and vegetables, such as celery tuber. We purified IgG/IgE antibodies from the serum of an exclusively profilin-allergic patient using affinity columns either coupled with protein extracts from mugwort pollen, birch pollen, or celery tuber. Constrained and unconstrained random nonapeptide libraries were pooled and screened with the anti-profilin antibody preparations to define cross-reactive ligands. Specific ligands were enriched by successive panning rounds using the profilin-specific antibodies in series. After the last panning round enriched phage clones were screened with purified profilin-specific antibodies and IgE-binding clones were sequenced. Five out of eight positive clones (62.5 %) displayed the same circular peptide CAISGGYPVC. This peptide was synthesized and examined for its ability to inhibit IgE binding to blotted mugwort pollen, birch pollen, or celery tuber profilin. Inhibition studies showed reduction of IgE binding to profilins in all three protein extracts. As the sequence of the mimotope did not show any homology to the known birch profilin sequence this peptide is considered to mimic a common conformational IgE epitope for these examined profilins.

Allergens↗
PubMed1998

Selection dynamics in autocatalytic systems: templates replicating through binary ligation.

The theory of autocatalytic binary ligation is reviewed within the context of a consistently applied Michaelis-Menten quasi-steady-state approximation to obtain explicit analytical results describing time-course data from experiments. A detailed protocol for the step-wise elucidation of a minimal set of experimental parameters is outlined. The kinetic equations are then generalized to cases of self- and cross-catalysis among an arbitrary number of different templates and applied to experiments involving just two templates. Depending on the values of various kinetic parameters such systems can display exclusionary Darwinian selection corresponding to an exponential growth law, selective coexistence or coexistence of all species characteristic of a parabolic growth law; the intermediate behaviour arises as a property of the full mechanism analysed here. Our results are applicable to the classical case of self-replicating nucleic acids and their analogues as well as to newly discovered self-replicating peptides.

Catalysis↗
PubMed1998

Recombinant human natural autoantibodies against GPIIb/IIIa inhibit binding of autoantibodies from patients with AITP.

Autoimmune thrombocytopenic purpura (AITP) is caused by autoantibodies predominantly against platelet membrane glycoproteins (GP) IIb/IIIa and GPIb/IX. Naturally occurring autoantibodies have been described against a variety of autoantigens; it has been suggested that perturbation of their regulation may be associated with autoimmune diseases. Using a combinatorial Fab phagemid library from an individual immunized with human RhD+ red blood cells, we evaluated the presence of natural anti-GPIIb/IIIa autoantibodies as well as their relation to AITP-associated anti-GPIIb/IIIa autoantibodies. Selection on native GPIIb/IIIa and characterization of positive clones by inhibition studies against murine monoclonal anti-GPIIb/IIIa antibodies and by DNA analysis revealed the presence of two distinct recombinant anti-GPIIb/IIIa autoantibodies, which partially inhibited binding of affinity-purified platelet-associated autoantibodies from 8/12 AITP patients. Our results demonstrated that GPIIb/IIIa-specific Fab directed against conformational epitopes within the GPIIb/IIIa complex may be cloned from the genome of an individual immunized with RhD+ red blood cells, who was not affected by AITP. The partial inhibition of binding of platelet-associated autoantibodies from AITP patients to GPIIb/IIIa by the recombinant anti-GPIIb/IIIa phage clones suggests recognition of closely related antigenic epitopes. These phage clones may represent down-regulated, potentially pathological autoantibodies and could be used as new tools for investigation of AITP.

Amino Acid Sequence↗
PubMed1998

Antigen interaction and heat inactivation expose new epitopes on human IgE.

It is well established that heat-denatured IgE is no longer capable of binding to FcepsilonRI. We have found an antibody that interacts with heat-denatured IgE. Interestingly, this antibody can also be used to detect some serum IgE, but not IgE synthesized de novo in vitro. However, native IgE can be transformed into an IgE that is recognized by this antibody, if antigen is added. Our data indicate that physiological mechanisms exist that biologically inactivate IgE which might still be mistaken for 'functional' IgE by assays based on polyclonal antibodies.

Animals↗
PubMed1998

Sequence and specificity analysis of recombinant human Fab anti-Rh D isolated by phage display.

BACKGROUND AND OBJECTIVES: Hyperimmune anti-Rh D serum is worldwide in short supply. As a first step to develop an alternative source of Rh D antibodies, we describe in this study the isolation and characterization of recombinant anti-Rh D Fab fragments. MATERIALS AND METHODS: Peripheral blood mononuclear cells harvested from a hyperimmunized donor were used to construct two combinatorial Fab libraries. Phages expressing these Fab fragments were selected on whole red blood cells followed by testing of positive clones in an indirect hemagglutination assay. RESULTS: Individual Fab clones are of high affinity and competitively inhibit the binding of a registered anti-D immunoglobulin. The Fab clones are also specific against the partial D phenotypes, Rh33, DIII, DIVa, DIVb, DVa, and DVII. The 13 different but highly homologous clones express preferentially VH3 segments. CONCLUSION: These Fab fragments show potential for the development of a new generation of therapeutic anti-Rh D reagents.

Amino Acid Sequence↗
PubMed1997

Nonspecific binding of IgE to allergens.

Nonspecific IgE binding to allergens was observed in testing myeloma IgEs, namely, IgE-VL and IgE-PS, chimeric IgE (IgE-JW8), and the recombinant IgE Fc epsilon peptide CH1-CH4, in two different immunoassays. This binding was concentration-dependent but detectable only at higher IgE concentration. In RAST inhibition, IgE-allergen interactions could be reduced by using either matching or nonmatching allergens. In order to test whether the nonspecific binding of IgE to allergens was due to carbohydrate interaction, myeloma IgEs and the chimeric IgE were desialized with neuraminidase. Desialized samples were equally well recognized by xenogenic antibodies as native IgEs, but binding of IgE to Fc epsilon receptors on basophils was affected by the treatment, as shown in the histamine-release assay. Desialization of IgE affected also its binding capacity to allergens in RAST: binding of chimeric IgE was reduced, but nonspecific binding of myeloma IgE-VL was enhanced. Hence, nonspecific allergen-IgE binding may be partly due to a lectin-like interaction, but may depend mostly on the tertiary structure of IgE. Thus, nonspecific IgE-allergen interactions might present a problem 1) at high IgE concentration, and 2) depend on the grade of sialization of IgE, which might affect its conformation. This may explain why patients with elevated total IgE levels often have multiple weak positive RASTs with non-cross-reactive allergens.

Allergens↗
PubMed1996

Effect of anti-IgE antibodies on Fc epsilonRI-bound IgE.

Anti-IgE mAbs have always been used to trigger mediator release from basophils or mast cells. Now nonanaphylactogenic anti-human IgE Abs are in clinical evaluation as a therapeutic agent against atopic disease. We have found a mAb that is nonanaphylactogenic but recognizes receptor-bound IgE. Interestingly, the Ab prevents the association of IgE with its receptor if immune complexes were formed between IgE and anti-IgE mAb. This explained the phenomenon that addition of anti-IgE Ab to receptor-bound IgE resulted in a decrease of receptor-bound IgE, because IgE dissociating from the receptor was complexed, altering the thermodynamic balance of receptor-bound vs free IgE. Our data show that there are nonanaphylactogenic Abs that do not directly interfere with the receptor binding site on IgE and are capable of preventing the association of IgE with its high affinity receptor. This unique feature would render such an anti-IgE Ab a possible candidate for immunotherapy of atopy.

Anaphylaxis↗
PubMed1996

Anti-IgE in allergic sensitization.

Anti-IgE autoantibodies exist predominantly in the sera of patients with atopic disease. For some time such anti-IgE autoantibodies have been considered a phenomenon that may not be of clinical importance. The cloning of such anti-IgE autoantibodies has eliminated doubts of whether these antibodies exist, but it is still unclear whether such autoantibodies play a pathophysiological role. However, there are ongoing clinical trials that use humanized anti-IgE antibodies for passive immunization of atopic individuals. While this approach may not definitely clarify the role of anti-IgE autoantibodies, it will nevertheless clarify the role of IgE.

Animals↗
PubMed1996

Neuropeptides accentuate interleukin-4 induced human immunoglobuline E synthesis in vitro.

Corticotropin releasing factor, adrenocorticotropic hormone (ACTH) and alpha-melanocyte stimulating hormone either inhibit or enhance in a dose-dependent fashion an interleukin-4 (IL-4) driven human IgE synthesis in vitro. Here, we show that culture conditions strongly influence the earlier observed dose- and donor-dependent effects of adrenocorticotropic hormone. The effect of ACTH on IgE synthesis became only apparent late during culture periods, suggesting an indirect effect via the cellular microenvironment rather than by acting directly at the level of B-cell isotype switching. Thus, we studied other proopiomelanocortin (POMC) derived peptides and neuropeptides known to influence the cellular microenvironment. Indeed, similar modulatory effects on IgE synthesis were also observed by the addition of other proopiomelanocortin-derived peptides such as alpha-, beta-, and gamma-endorphins as well as by the opioid binding pentapeptide Leu-enkephalin. Furthermore the neuropeptide substance P accentuated an IL-4 or an IL-4 and anti-CD40 antibody driven class switch to IgE. In contrast to ACTH, substance P interfered not only with IgE synthesis but also with the synthesis of the other immunoglobulin isotypes. Thus, systemically acting neuroendocrine peptides such as ACTH and locally acting neuropeptides such as the enkephalins and substance P can modulate the magnitude of an IL-4 induced IgE response.

Adrenocorticotropic Hormone↗
PubMed1996

Basophil histamine release and leukotriene production in response to anti-IgE and anti-IgE receptor antibodies. Comparison of normal subjects and patients with urticaria, atopic dermatitis or bronchial asthma.

The IgE receptor-dependent in vitro mediator release in basophils is characterized by a large interindividual variability both in normal and atopic subjects. The mechanism and the clinical impact of this finding, however, is largely unclear. The aim of the present study was to examine the role of surface-bound IgE and of response-modifying cytokines such as interleukin 3 (IL-3) as possible factors determining basophil releasability in atopic patients and normal controls. Cells from 30 individuals (6 with urticaria, 7 with asthma, 7 with atopic dermatitis, and 30 healthy controls) were isolated and stimulated for mediator release by IL-3 and different triggering antibodies directed against IgE or IgE receptor. Our data suggest that serum IgE levels and basophil receptor occupancy with IgE are not involved in the mechanism of basophil releasability. Furthermore, IL-3-induced similar effects on mediator release in almost all individuals, rather excluding the possibility that releasability is regulated by cytokine priming of basophils. Interestingly, we found that patients with atopic disease have a reduced capacity of releasing mediators upon activation, the mechanism of which is unclear. In conclusion, our findings support the hypothesis that basophil releasability is dependent on cell-imminent mechanisms in basophils, which may be altered in selected atopic patients.

Adult↗
PubMed1996

Culture and IgE synthesis of nasal B cells.

The interplay of inflammatory cells, mediators and cytokines during type I allergic reactions in the nose is well described. But even though allergen-specific IgE is known to play a central role for the induction of these events, little is known about nasal B cells and their role in the local allergic reaction. It was the aim of the present study to examine the possibility to isolate and culture B cells from the nose. For this purpose allergic and nonallergic volunteers were challenged by nasal provocation with allergen. Cells were collected sequentially after nasal provocation by nasal lavage, B cells grown in the CD40 system and IgE production assessed by enzyme-linked filter spot assay and radioimmunoassay. IgE-producing B cells were detected after culture of nasal lavage cells in the CD40 system. Elevated levels of IgE were measured in culture supernatants from cells collected during the late-phase allergic reaction. We conclude that B cells can be isolated from the nose and may serve as an interesting source of B cells after in vivo contact with antigen.

B-Lymphocytes↗
PubMed1996

Cross reactive anti-tetanus and anti-melittin Fab fragments by phage display after tetanus toxoid immunisation.

Amino acid sequence homology between tetanus toxoid, a common vaccine and melittin, a bee venom allergen provided us with two antigen models for studying the production of cross reactive antibodies after immunisation. Analysis of the serum of an atopic patient recently boosted with tetanus toxoid revealed the presence of anti-melittin and anti-tetanus antibodies. From this donor a phage display Fab library was constructed five days after vaccination. Screening of this library either with melittin alone or with tetanus toxoid followed by melittin allowed the isolation of Fab fragments specific to melittin but also Fab fragments which cross react with melittin and tetanus toxoid. Amino acid analysis revealed diversity in the heavy and the light Ig chains of the melittin specific and of the cross reactive clones. Interestingly we found that the light chain recognised melittin whereas the heavy chain preferentially bound to tetanus toxoid suggesting that cross reactivity may be due to the different binding specificities of the individual Ig chains.

Amino Acid Sequence↗
PubMed1995

Antigen-specific inhibition of IgE binding to the high-affinity receptor.

Since the beginning of this century, allergen immunotherapy has been widely used to treat allergic disorders. In addition to the long-term clinical efficacy of this therapy, there are immediate beneficial effects as observed in rush immunotherapy for which there is no clear mechanism. We investigated the direct impact of an Ag on its specific IgE in terms of IgE measurability in immunoassays and subsequent binding of IgE to the high-affinity receptor for IgE. As a model we used a chimeric IgE specific for NIP that exhibits similar biologic properties as serum or myeloma IgE. To mimic particulate and soluble allergens we coupled 15 NIP molecules to BSA. Using this "artificial allergen" we could show that the presence of the Ag reduced the IgE measurability in immune assays. Furthermore, IgE binding to the Fc epsilon RI was 60% inhibited in the presence of the Ag shown with an optical biosensor that monitors molecular interactions. In normal basophils passively sensitized with IgE that was preincubated with the Ag, no sulfidoleukotriene release could be induced. Rush immunotherapy may invoke a similar phenomenon, resulting in the short-term alteration of symptoms by blocking or substantially reducing binding of IgE to its high-affinity receptor. Thus, our result may explain some of the short-term beneficial effects observed in rush immunotherapy.

Allergens↗