Biomedical subjects

Beda M Stadler

Publications and source records attributed to Beda M Stadler.

12 recordsLinked to original sources

PubMed2006

Immunologic and functional evidence for anti-Siglec-9 autoantibodies in intravenous immunoglobulin preparations.

Human intravenous immunoglobulin (IVIg) preparations are increasingly used for the treatment of autoimmune diseases. Earlier work demonstrated the presence of autoantibodies against Fas in IVIg, suggesting that IVIg might be able to induce caspase-dependent cell death in Fas-sensitive cells. In this study, we demonstrate that sialic acid-binding Ig-like lectin 9 (Siglec) represents a surface molecule on neutrophils that is activated by IVIg, resulting in caspase-dependent and caspase-independent forms of cell death. Neutrophil death was mediated by naturally occurring anti-Siglec-9 autoantibodies present in IVIg. Moreover, the efficacy of IVIg-mediated neutrophil killing was enhanced by the proinflammatory cytokines granulocyte/macrophage colony-stimulating factor (GM-CSF) and interferon-gamma (IFN-gamma), and this additional cell death required reactive oxygen species (ROSs) but not caspases. Anti- Siglec-9 autoantibody-depleted IVIg failed to induce this caspase-independent neutrophil death. These findings contribute to our understanding of how IVIg preparations exert their immunoregulatory effects under pathologic conditions and may provide a possible explanation for the neutropenia that is sometimes seen in association with IVIg therapy.

Antigens, CD↗

PubMed2006

Internal images: human anti-idiotypic Fab antibodies mimic the IgE epitopes of grass pollen allergen Phl p 5a.

BACKGROUND: The role of anti-idiotypic antibodies in allergic disease is still poorly understood. According to Jerne, anti-idiotypic antibodies to IgE should represent internal images of an allergen. Our aim was to ultimately prove whether this hypothesis holds true in allergy. Here, we describe the selection of anti-idiotypic antibodies against Phl p 5a-specific IgE directly from the B-cell repertoire of a grass pollen allergic individual. METHODS: Taking Phleum pratense grass pollen allergen Phl p 5 as a model, we selected anti-idiotypic antibodies against allergen-specific IgE directly from the B-cell repertoire of an allergic individual. We screened a combinatorial phage display library of human monovalent antibody heavy and light chain fragments (Fabs) with anti-Phl p 5a-IgE to identify and characterize Fabs with anti-idiotypic specificity. RESULTS: Five different Fab clones with anti-idiotypic specificity for anti-Phl p 5a-IgE were identified. Their hypervariable regions revealed partial sequence homology with solvent accessible antigenic sites of Phl p 5a, which have been identified by our previous mimotope approach. Phagemid DNA derived from the phage clones was used to produce two soluble recombinant anti-idiotypic Fab clones in E. coli. As a proof of molecular mimicry, both Fabs induced anti-Phl p 5a-specific antibodies in immunized BALB/c mice. Molecular modeling of the heavy and light chain hypervariable loops of the anti-idiotypic Fabs illustrated structural similarity with dominant IgE epitopes of Phl p 5a. CONCLUSION: In this straightforward phage technology approach, antibodies with anti-idiotypic specificities could be isolated from a human allergic's repertoire. As predicted by the immune network hypothesis, their hypervariable domains mimic IgE epitopes like internal images and, more importantly, induce allergen-specific immune responses in the absence of the allergen.

Amino Acid Sequence↗

PubMed2006

In silico predictability of allergenicity: from amino acid sequence via 3-D structure to allergenicity.

In relation to the prediction of allergenicity three aspects have to be discussed: IgE immunogenicity, IgE cross-reactivity, and T-cell cross-reactivity. IgE immunogenicity depends largely on factors other than the protein itself: the context and dose and "history" of the protein by the time it reaches the immune system. It is, therefore, not fully predictable from structural information. In contrast, IgE cross-reactivity can be much more reliably assessed by in-silico homology searches in combination with in vitro IgE antibody assays. The in-silico homology search is unlikely to miss potential cross-reactivity with sequenced allergens. So far, no biologically relevant cross-reactivity at the antibody level has been demonstrated between proteins without easily demonstrable homology. T-cell cross-reactivity is much more difficult to predict than B-cell cross-reactivity. Moreover, its effects are more diverse. Yet, pre-existing cross-reactive T-cell activity is likely to influence the outcome not only of the immune response, but also of the effect phase of the allergic reaction. The question of whether any antigen can be allergenic is still a matter of debate.

Allergens↗

PubMed2005

A high-affinity natural autoantibody from human cord blood defines a physiologically relevant epitope on the FcepsilonRIalpha.

Natural Abs represent the indigenous immune repertoire and are thus present at birth and persist throughout life. Previously, human autoantibodies to the alpha domain of the high-affinity IgE receptor (FcepsilonRIalpha) have been isolated from Ab libraries derived from normal donors and patients with chronic urticaria. To investigate whether these anti-FcepsilonRIalpha Abs are present in the germline repertoire, we constructed a phage Fab display library from human cord blood, which represents the naive immune repertoire before exposure to exogenous Ags. All isolated clones specific to the FcepsilonRIalpha had the same sequence. This single IgM Ab, named CBMalpha8, was strictly in germline configuration and had high affinity and functional in vitro anaphylactogenic activity. Inhibition experiments indicated an overlapping epitope on the FcepsilonRIalpha recognized by both CBMalpha8 and the previously isolated anti-FcepsilonRIalpha Abs from autoimmune and healthy donors. This common epitope on FcepsilonRIalpha coincides with the binding site for IgE. Affinity measurements demonstrated the presence of Abs showing CBMalpha8-like specificity, but with a significantly lower affinity in i.v. Ig, a therapeutic multidonor IgG preparation. We propose a hypothesis of escape mutants, whereby the resulting lower affinity IgG anti-FcepsilonRIalpha Abs are rendered less likely to compete with IgE for binding to FcepsilonRIalpha.

Amino Acid Sequence↗

PubMed2005

Detection of one VH antibody sequence in both healthy donors and urticaria patients.

We have previously isolated anti-FcepsilonRIalpha autoantibodies from phage libraries of healthy donors and urticaria patients. Strikingly, the same antibody, LTMalpha15, was isolated from both libraries. Sequence analysis revealed a germline configuration of the LTMalpha15 variable heavy (V(H)) chain with a slightly mutated variable light (V(L)) chain supporting its classification as a natural autoantibody. Distribution analysis of anti-FcepsilonRIalpha autoantibodies by functional or serological tests delivered conflicting data. For this reason we have developed a new real-time PCR to analyse the distribution of LTMalpha15V(H) in healthy donors and urticaria patients. Our new bioinformatic program permitted the design of a minor groove binder (MGB) TaqMan probe that specifically detected the LTMalpha15V(H). We were able to demonstrate a broad range of rearranged V(H) gene copy number without any correlation to the state of health. Monitoring LTMalpha15V(H) gene copy number in a single donor over a period of 70 days revealed a time-related fluctuation of circulating B cells carrying LTMalpha15V(H). We propose that our real-time PCR may serve as a model for the quantification of natural antibody sequences at a monoclonal level.

Autoantibodies↗

PubMed2005

Intranasal immunisation using recombinant Lactobacillus johnsonii as a new strategy to prevent allergic disease.

We have previously demonstrated the induction of a specific anti-IgE response in vivo by parenteral immunisation of rhesus monkeys using short IgE mimotopes or an anti-idiotypic antibody mimicking an IgE epitope. Such specific anti-IgE responses may be of clinical benefit for atopic patients. In this study, we examined the potential for a more convenient therapy via mucosal immunisation using live recombinant Lactobacillus johnsonii (Lj) as a vaccine delivery vehicle. Either an anti-idiotypic scFv or an IgE mimotope were expressed on the surface of Lj as fusion proteins using the cell wall anchored proteinase PrtB from Lactobacillus delbrueckii subsp. bulgaricus. The recombinant Lj were shown to express the heterologous fusion proteins and were specifically recognised by the corresponding anti-human IgE monoclonal antibody. Subcutaneous and intranasal immunisation of mice with recombinant Lj, expressing these fusion proteins induced a systemic IgG response against human IgE. Our data suggest that recombinant Lactobacilli expressing IgE epitopes may represent a novel means of vaccination to induce a beneficial anti-IgE response.

Amino Acid Sequence↗

PubMed2005

Single nucleotide polymorphisms as a prerequisite for autoantigens.

It is still elusive why certain self proteins induce an autoimmune response. One immunological hypothesis is that only modified or altered self-proteins may become a target. Thus, we asked whether such alterations may actually be genetic polymorphisms that can be revealed by analyzing sequence variability in the known human autoantigens. Indeed, we found autoantigens to contain significantly more single nucleotide polymorphisms (SNP) than other human genes do. Our finding may offer an explanation for autoimmune responses through allogeneic exposure. Besides other contributing factors in autoimmunity, SNP may represent an essential prerequisite for the primary induction of an autoimmune response.

Amino Acid Sequence↗

PubMed2004

A highly conserved interspecies V(H) in the human genome.

Idiotype conservation between human and mouse antibodies has been observed in association with various infectious and autoimmune diseases. We have isolated a human anti-idiotypic antibody to a mouse monoclonal anti-IgE antibody (BSW17) suggesting a conserved interspecies idiotype associated with an anti-IgE response. To find the homologue of BSW17 in the human genome we applied the guided selection strategy. Combining V(H) of BSW17 with a human V(L) repertoire resulted in three light chains. The three V(L) chains were then combined with a human V(H) repertoire resulting in three clones specific for human IgE. Surprisingly, one clone, Hu41, had the same epitope specificity and functional in vitro activity as BSW17 and V(H) complementarity-determining regions identical with that of BSW17. Real-time PCR analysis confirmed the presence of the Hu41 V(H) sequence in the human genome. These data document the first example of the isolation of a human antibody where high sequence similarity to the original murine V(H) sequence is associated with common antigen and epitope specificity.

Amino Acid Sequence↗

PubMed2004

Natural anti-FcepsilonRIalpha autoantibodies may interfere with diagnostic tests for autoimmune urticaria.

IgG autoantibodies against the alpha-chain of the high affinity IgE receptor are claimed to play a pathogenetic role in autoimmune urticaria. The best methods for detection of functional autoantibodies are currently the autologous serum skin test and the basophil histamine release assay. A simplified and feasible screening test would facilitate the diagnosis of autoimmune urticaria. Here we offer an explanation for the difficulties in establishing a screening test for autoantibodies directed against the alpha-chain of the high affinity IgE receptor in autoimmune urticaria. Identical autoantibodies in chronic urticaria patients and healthy donors belonging to the natural autoantibody repertoire were found by sequence analysis of anti-alpha-chain autoantibodies isolated by repertoire cloning from antibody libraries. These natural autoantibodies bound to the receptor and triggered histamine release but only if IgE was previously removed from the receptor. Diagnostic assays used for detection of antibodies directed against the IgE receptor may require signal comparison with and without the artificial removal of IgE, immune complexes, and complement in order to avoid false positive or negative results. After IgE removal diagnostic tests will detect natural autoantibodies against the high affinity IgE receptor regardless of whether they are pathogenic or not.

Amino Acid Sequence↗

PubMed2003

Allergenicity prediction by protein sequence.

Potential allergenicity of transgenic proteins for consumption must be investigated before their introduction into the food chain. A prerequisite is sequence analysis. We have critically reviewed the performance of the current guidelines proposed by the Food and Agriculture Organization (FAO) and the World Health Organization (WHO) for allergenicity prediction based on protein sequence and show that its precision is very low. To improve prediction, we propose a new strategy based on sequence motifs identified from a new allergen database. If tested on random test sequences and known allergens, both methods are apparently very sensitive. However, the precision of our motif-based prediction (95.5%) is superior to the current method (36.6%). We conclude that the proposed motif-based prediction is a superior alternative to the current method for use in the decision-tree approach for allergenicity assessment.

Allergens↗

PubMed2002

Recombinant Lactobacillus johnsonii as a mucosal vaccine delivery vehicle.

Lactobacilli are considered to be safe organisms making them attractive as vehicles for oral vaccination. We report that Lactobacillus johnsonii (Lj) partially survived simulated gastric conditions in vitro, suggesting that it could be used as an oral vaccine delivery vehicle. In order to test this approach, we used the cell wall anchored proteinase PrtB, isolated from Lactobacillus delbrueckii subsp. bulgaricus as a model antigen. Using a new vector system, we demonstrated expression of both proteinase PrtB alone and a mimotope peptide derived from tetanus toxin integrated in the sequence of proteinase PrtB (TTmim-PrtB fusion protein) on the surface of Lj. Oral immunisation of mice with recombinant Lj, expressing the TTmim-PrtB fusion protein induced a systemic IgG response against Lj and recombinantly expressed proteinase PrtB but no antibody response against the tetanus toxin mimotope suggesting that the mimotope was not sufficiently immunogenic to induce an immune response. Interestingly, a proteinase PrtB specific fecal IgA response was also induced, indicating that the proteinase PrtB fusion protein expressed as a cell surface protein on Lj can induce both systemic and local mucosal immune responses.

Administration, Oral↗

PubMed2002

Scavenger receptor block as strategy for the identification of bone marrow homing phages by panning in vivo random peptide phage displayed libraries.

Surface molecules exclusively expressed by cells of the bone marrow (BM) are candidate targets for delivering therapeutic agents to this tissue. To identify ligands specific for the BM, we performed a series of pannings in vivo with random peptide phage displayed libraries (RPPDL). We could show that phages bind to bone marrow endothelium (BME) independently of the peptide insert, suggesting that the BM, similarly to spleen and liver, is part of the reticulo-endothelial system (RES). Furthermore, this strong "natural" affinity to the BME was abrogated by polyanions, indicating that phage trapping by this endothelium is mediated by scavenger receptors (SR). To circumvent interference by SR, polyinosinic acid was administered before phage panning in vivo. This led to the identification of a consensus motif that confers binding specificity for a subpopulation of hemopoietic marrow cells. Thus, SR inhibition, by avoiding phage trapping by the endothelium, seems to allow phage particles to extravasate and reach parenchymal cells. Accordingly, this panning strategy in vivo may be useful for the identification of targeting motifs specific for cells located in the extravascular space of various tissues.

Amino Acid Motifs↗