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Biomedical subjects

B M Stadler

Publications and source records attributed to B M Stadler.

At least 19 recordsLinked to original sources
PubMed2002

Recombinant anti-idiotypic antibodies inhibit human natural anti-glycoprotein (GP)IIb/IIIa autoantibodies.

Anti-idiotypic antibodies (anti-Id) have been described against idiotypes expressed on various autoantibodies. Since an immunoregulatory effect has been postulated for anti-Id, modulation of the anti-Id response in autoimmune disease may be of interest. In chronic immune thrombocytopenic purpura (AITP), autoantibodies directed mainly against platelet membrane glycoprotein (GP) IIb/IIIa cause platelet destruction by Fc-mediated phagocytosis or by complement lysis. We have previously reported on the generation of two recombinant anti-GPIIb/IIIa autoantibody fragments (PDG-X, PDG-B), that are specific for conformationally intact GPIIb/IIIa and inhibit binding of autoantibodies from patients with AITP. In the present study, we show that anti-GPIIb/IIIa specificities are not limited to a single individual by isolating five additional anti-GPIIb/IIIa autoantibody fragments from a second phagemid Fab library of an unrelated healthy donor. Using soluble Fab of PDG-X and PDG-B as antigens for panning Fab phagemid libraries from healthy human individuals, we isolated anti-Id phage clones specific for PDG-X or PDG-B. In addition they inhibited the binding of PDG-X or PDG-B to GPIIb/IIIa. Amino acid sequence comparison between these specific antiId and GPIIb/IIIa was performed. Generation of these anti-Id directed against pathologically relevant anti-GPIIb/IIIa autoantibodies may represent a new suitable and specific therapeutic option for the treatment of antibody-mediated AITP.

Amino Acid Sequence↗
PubMed2001

The topology of the possible: formal spaces underlying patterns of evolutionary change.

The current implementation of the Neo-Darwinian model of evolution typically assumes that the set of possible phenotypes is organized into a highly symmetric and regular space equipped with a notion of distance, for example, a Euclidean vector space. Recent computational work on a biophysical genotype-phenotype model based on the folding of RNA sequences into secondary structures suggests a rather different picture. If phenotypes are organized according to genetic accessibility, the resulting space lacks a metric and is formalized by an unfamiliar structure, known as a pre-topology. Patterns of phenotypic evolution-such as punctuation, irreversibility, modularity--result naturally from the properties of this space. The classical framework, however, addresses these patterns by exclusively invoking natural selection on suitably imposed fitness landscapes. We propose to extend the explanatory level for phenotypic evolution from fitness considerations alone to include the topological structure of phenotype space as induced by the genotype-phenotype map. We introduce the mathematical concepts and tools necessary to formalize the notion of accessibility pre-topology relative to which we can speak of continuity in the genotype-phenotype map and in evolutionary trajectories. We connect the factorization of a pre-topology into a product space with the notion of phenotypic character and derive a condition for factorization. Based on anecdotal evidence from the RNA model, we conjecture that this condition is not globally fulfilled, but rather confined to regions where the genotype-phenotype map is continuous. Equivalently, local regions of genotype space on which the map is discontinuous are associated with the loss of character autonomy. This is consistent with the importance of these regions for phenotypic innovation. The intention of the present paper is to offer a perspective, a framework to implement this perspective, and a few results illustrating how this framework can be put to work. The RNA case is used as an example throughout the text.

Animals↗
PubMed2001

Conditional autoantibodies in urticaria patients: a unifying hypothesis.

Chronic urticaria may be characterized by conditional autoantibodies against the alpha-chain of the high-affinity receptor for IgE (FcepsilonRI). These autoantibodies are termed conditional as they only recognize unoccupied FcepsilonRI. The same conditional reactivity pattern has also been found in sera of atopic and normal healthy donors. Any condition resulting in accessibility of FcepsilonRI will render these autoantibodies anaphylactogenic. This finding offers a unifying hypothesis for the manifestation of different forms of urticaria. Non-immunologic triggers may thereby influence directly or indirectly the number of accessible FcepsilonRI allowing the conditional autoantibodies to induce urticaria symptoms.

Autoantibodies↗
PubMed2001

Conditional autoimmunity mediated by human natural anti-Fc(epsilon)RIalpha autoantibodies?

Natural antibodies provide an early defense mechanism against pathogens, show a frequent self-reactivity, and are present throughout life. Two questions concern the physiological control of self-reactivity and the pathogenetic link to autoimmune disease. Here we propose a concept of conditional autoimmunity involving natural antibodies against the alpha chain of the high-affinity receptor for IgE (Fc(epsilon)RIalpha ). Like other natural antibodies, anti-Fc(epsilon)RIalpha antibodies are found in sera of healthy donors. We now report the first human recombinant anti-Fc(epsilon)RIalpha autoantibodies isolated by repertoire cloning from a human tonsillar IgM library. These high-affinity antibodies recognize Fc(epsilon)RIalpha on cells and trigger histamine release from freshly isolated blood basophils. However, the latter effect requires IgE removal from the Fc(epsilon)RI. The same conditional histamine release is seen when using sera from individual normal donors and affinity-purified anti-Fc(epsilon)RIalpha antibodies isolated from multidonor therapeutic IgG preparations. We propose that such anti-Fc(epsilon)RIalpha antibodies can become pathogenic and that this is dependent on the state of occupancy of the Fc(epsilon)RIalpha by its natural ligand IgE. We suggest that an imbalance between Fc(epsilon)RIalpha occupancy and natural anti-Fc(epsilon)RIalpha antibodies may be implicated in the pathogenesis of autoimmune urticaria.

Amino Acid Sequence↗
PubMed2001

Natural anti-FcepsilonRIalpha autoantibodies isolated from healthy donors and chronic idiopathic urticaria patients reveal a restricted repertoire and autoreactivity on human basophils.

The role of autoantibodies against the alpha-subunit of the human high-affinity IgE receptor (FcepsilonRIalpha) in the pathogenesis of chronic idiopathic urticaria (CIU) is controversial. We have shown that these antibodies are widespread, apparently non-pathogenic and belong to the natural antibody repertoire. To clarify this controversy, we constructed antibody libraries from both healthy donors and CIU patients with active disease. Here we describe the first three high affinity IgM anti-FcepsilonRIalphaautoantibodies isolated from normal and urticaria libraries. Sequence analysis revealed germline VH in both cases paired with a slightly mutated VL, thus supporting their classification as natural antibodies. Strikingly, one major IgM clone was present in both CIU patients and normal donors. The anti-FcepsilonRIalpha autoantibodies recognize FcepsilonRIalpha on cells, but are non-anaphylactogenic on blood basophils, except when IgE is removed from the receptor. Based on their functional activities we propose a concept of "conditional autoimmunity" where natural anti-FcepsilonRIalphaautoantibodies can become pathogenic dependent on the state of occupancy of the FcepsilonRIalpha by its natural ligand IgE.

Animals↗
PubMed2000

Molecular basis for nonanaphylactogenicity of a monoclonal anti-IgE antibody.

IgE Abs mediate allergic responses by binding to specific high affinity receptors (FcepsilonRI) on mast cells and basophils. Therefore, the IgE/FcepsilonRI interaction is a target for clinical intervention in allergic disease. An anti-IgE mAb, termed BSW17, is nonanaphylactogenic, although recognizing IgE bound to FcepsilonRI, and interferes with binding of IgE to FcepsilonRI. Thus, BSW17 represents a candidate Ab for treatment of IgE-mediated disorders. By panning BSW17 against random peptide libraries displayed on phages, we defined mimotopes that mimic the conformational epitope recognized on human IgE. Two types of mimotopes, one within the Cepsilon3 and one within the Cepsilon4 domain, were identified, indicating that this mAb may recognize either a large conformational epitope or eventually two distinct epitopes on IgE. On the basis of alignments of the two mimotopes with the human IgE sequence, we postulate that binding of BSW17 to the Cepsilon3 region predominantly blocks binding of IgE to FcepsilonRI, leading to neutralization of IgE. Moreover, binding of BSW17 to the Cepsilon4 region may explain how BSW17 recognizes FcepsilonRI-bound IgE, and binding to this region may also interfere with degranulation of IgE sensitized cells (basophils and mast cells). As a practical application of these findings, mimotope peptides coupled to a carrier protein may be used for the development of a peptide-based anti-allergy vaccine by induction of anti-IgE Abs similar to the current approach of using humanized nonanaphylactogenic anti-IgE Abs as a passive vaccine.

Amino Acid Sequence↗
PubMed2000

Mimicry of human IgE epitopes by anti-idiotypic antibodies.

According to Jerne's network hypothesis, the binding site of an anti-idiotypic antibody also represents the internal image of an epitope present on a foreign, or even a self antigen. In recent years, antigen mimicry has been defined at the molecular level for some xeno-antigens. However, until now there has been no demonstration of structural mimicry between a human anti-idiotypic antibody and a self structure. To address this question, we used human IgE as the self structure and a well-defined anti-human IgE mAb (BSW17). We describe the isolation of two anti- idiotypic antibodies specific for the anti-IgE antibody BSW17 from a non-immune human Fab phage display library. Interestingly, these two anti-idiotypic antibodies mimic the same molecular surface region as a previously described IgE peptide mimotope isolated by panning on BSW17, but they cover a much larger epitope on the IgE molecule. Accordingly, immunisation of rabbits with the two anti-idiotypic antibodies induced high-affinity antibodies with the same characteristics as BSW17. Thus, our data demonstrate that it is possible to isolate anti-idiotypic antibodies derived from the human genome without the need for hyperimmunization, and confirm Jerne's hypothesis that both foreign antigens and self structures can be mimicked by our own immunoglobulins.

Amino Acid Sequence↗
PubMed2000

Oral anti-IgE immunization with epitope-displaying phage.

An essential requirement for oral vaccines is the ability to survive the harsh environment of the stomach in an antigenically intact form. As bacteriophages are adapted to this environment we used epitope-displaying M13 bacteriophages as carriers for an experimental oral anti-IgE vaccine. The feasibility of this approach was tested in a simulated gastric fluid using two different mimotopes as well as an anti-idiotypic Fab of the non-anaphylactogenic monoclonal anti-IgE antibody BSW17. All phage clones remained infective after this treatment. However, only epitopes displayed on the pVIII protein were still recognized by BSW17 whereas pIII-expressed epitopes were rapidly inactivated. Surprisingly, when used for oral immunization of mice all phage clones induced anti-IgE antibodies. In contrast, oral immunization with the purified, pVIII protein displaying the mimotope induced anti-phage but no anti-IgE antibodies. After feeding a single dose of mimotope-displaying bacteriophage, phage DNA could be detected in mouse feces for 10 days. Our results show that epitope-displaying bacteriophages can be used to induce an epitope-specific antibody response via the oral route.

Administration, Oral↗
PubMed2000

Dynamics of autocatalytic replicator networks based on higher-order ligation reactions.

A class of autocatalytic reaction networks based on template-dependent ligation and higher-order catalysis is analysed. Apart from an irreversible ligation reaction we consider only reversible aggregation steps that provide a realistic description of molecular recognition. The overall dynamics can be understood by means of replicator equations with highly non-linear interaction functions. The dynamics depends crucially on the total concentration c0 of replicating material. For small c0, in the hyperbolic growth regime, we recover the familiar dynamics of second-order replicator equations with its wealth of complex dynamics ranging from multi-stability to periodic and strange attractors as well as to heteroclinic orbits. For large c0, in the parabolic growth regime, product inhibition becomes dominating and we observe a single globally stable equilibrium tantamount to permanent coexistence. In an intermediate parameter range we sometimes observe a behavior that is reminiscent of 'survival of the fittest'. Independently replicating species (Schlögl's model) and the hypercycle are discussed in detail.

Catalysis↗
PubMed2000

CHO expression of a novel human recombinant IgG1 anti-RhD antibody isolated by phage display.

Replacement of the hyperimmune anti-Rhesus (Rh) D immunoglobulin, currently used to prevent haemolytic disease of the newborn, by fully recombinant human anti-RhD antibodies would solve the current logistic problems associated with supply and demand. The combination of phage display repertoire cloning with precise selection procedures enables isolation of specific genes that can then be inserted into mammalian expression systems allowing production of large quantities of recombinant human proteins. With the aim of selecting high-affinity anti-RhD antibodies, two human Fab libraries were constructed from a hyperimmune donor. Use of a new phage panning procedure involving bromelin-treated red blood cells enabled the isolation of two high-affinity Fab-expressing phage clones. LD-6-3 and LD-6-33, specific for RhD. These showed a novel reaction pattern by recognizing the D variants D(III), D(IVa), D(IVb), D(Va), D(VI) types I and II. D(VII), Rh33 and DFR. Full-length immunoglobulin molecules were constructed by cloning the variable regions into expression vectors containing genomic DNA encoding the immunoglobulin constant regions. We describe the first, stable, suspension growth-adapted Chinese hamster ovary (CHO) cell line producing a high affinity recombinant human IgG1 anti-RhD antibody adapted to pilot-scale production. Evaluation of the Fc region of this recombinant antibody by either chemiluminescence or antibody-dependent cell cytotoxicity (ADCC) assays demonstrated macrophage activation and lysis of red blood cells by human lymphocytes. A consistent source of recombinant human anti-RhD immunoglobulin produced by CHO cells is expected to meet the stringent safety and regulatory requirements for prophylactic application.

Animals↗
PubMed1999

Human anti-FcepsilonRIalpha autoantibodies isolated from healthy donors cross-react with tetanus toxoid.

Natural antibodies (Ab) reacting with self antigens have been shown to be present in all individuals. These autoantibodies (auto-Ab) can be either pathogenic or non-pathogenic. Auto-Ab reacting with the alpha-subunit of the high-affinity receptor for IgE (FcepsilonRIalpha) have been implicated in the pathogenesis of a subset of patients with chronic idiopathic urticaria (CIU). Intravenous immunoglobulin (IVIg) preparations have been used with variable clinical benefit in the treatment of these patients. Here we show that anti-FcepsilonRIalpha auto-Ab are present in a therapeutic IVIg preparation as well as in atopic and chronic urticaria patients and healthy individuals. We affinity-purified the anti-FcepsilonRIalpha Ab from an IVIg preparation using recombinant FcepsilonRIalpha. Interestingly, these anti-FcepsilonRIalpha auto-Ab showed no evidence of histamine release but strongly cross-reacted with an external antigen, tetanus toxoid (TTd) with a higher affinity for TTd than for the FcepsilonRIalpha. Since the cross-reacting Ab are non-anaphylactogenic, there is no evidence that TTd immunization may contribute to the pathogenesis of CIU. However, our results may indicate that the anti-FcepsilonRIalpha auto-Ab belong to the natural Ab and serve as the parental Ab for some anti-TTd Ab.

Animals↗
PubMed1999

Molecular mimicry of the unidentified antigen of myeloma antibody IgE-ND.

Antigen-specific human IgE is in short supply. Thus, we sought to determine the yet unknown specificity of a widely available human IgE, namely the myeloma cell line U266-derived IgE-ND. For this purpose highly specific peptides able to mimic the putative antigen recognized by IgE-ND were isolated from phage-display random peptide libraries. Interestingly, we found linear sequence homologies of the IgE-ND-binding peptides with self antigens and a xenoantigen from Thiobacillus ferrooxidans. However, none of these antigens was recognized by IgE-ND. Nevertheless, our approach may be applied to identify antigen specificities of myeloma antibodies. Importantly, the mimotopes were anaphylactogenic in a histamine release assay using human basophils sensitized with IgE-ND. Thus, our mimotopes represent functional albeit synthetic antigens and may be used to study human antigen-specific IgE responses.

Amino Acid Sequence↗
PubMed1999

High IgE in rheumatoid arthritis (RA) patients is complexed with anti-IgE autoantibodies.

This study presents data on more than 300 RA and allergic patients analysed for their serum levels of anti-immunoglobulin isotype autoantibodies and IgE. We observed high levels of IgE in sera of RA and allergic patients. Interestingly, we measured significantly higher specific IgE levels against Alternaria but not against nine other allergens in the RA compared with the allergic group. As expected, anti-IgG autoantibodies (rheumatoid factors (RF)) of different isotypes were detected in sera from RA patients only. However, we found increased titres of complexed anti-IgE autoantibodies in all RF+ groups and in the allergic group. These findings may explain why despite elevated IgE levels a decreased prevalence of allergic diseases in RA patients has been observed.

Adult↗
PubMed1999

Allergen mimotopes in food enhance type I allergic reactions in mice.

BIP1is a murine IgG antibody capable of enhancing the IgE binding to Bet v 1, the major birch pollen allergen. We have previously generated a mimotope of BIP1, designated Bet mim 1, from a constrained phage display peptide library. We demonstrated that oral immunization of BALB/c mice with the Bet mim 1 mimotope resulted in the induction of Bet v 1-specific IgG. The aim of this study was to test the influence of such an oral immunization with Bet mim 1 on a subsequent type I allergic response to Bet v 1. Phages displaying Bet mim 1 or control mimotopes, or PBS alone, were delivered to BALB/c mice by intragastric gavages prior to systemic sensitization with recombinant Bet v 1 and Al(OH)(3), an adjuvant inducing preferentially IgE antibody responses. Only mice fed with Bet mim 1-phages displayed substantially enhanced type I allergic skin reactivity to Bet v 1, as compared to mice pretreated with control mimotopes or PBS. A gastric digestion assay indicated that Bet v 1 and its homologue from apple, Mal d 1, were degraded within seconds under physiological conditions. In contrast, phage-displayed mimotopes were resistant to digestion. Our data indicate that allergen mimics in the diet that resist digestion, can induce allergen specific IgG able to enhance an allergic response. We therefore conclude that sensitization via the oral route may represent a mechanism for aggravating type I allergic reactions, probably leading to an earlier onset of symptoms even at lower allergen dosage.

Allergens↗
PubMed1999

Interaction of human IgE with Fc epsilon RI alpha exposes hidden epitopes on IgE.

BACKGROUND: Binding of human IgE via the heavy-chain constant region domain 3 (Cepsilon3) to the alpha-chain of its high affinity receptor (FcepsilonRIalpha) is a key event in mediating allergic reactions. We wanted to identify epitopes within Cepsilon3 that are stable to denaturation and to evaluate whether such structures are involved in receptor binding. The existence of stable epitopes would facilitate the generation of compounds that inhibit the IgE-FcepsilonRIalpha interaction. METHODS: Monoclonal anti-human IgE-antibodies against recombinant bacterially synthesized Cepsilon3, which is known to be partly misfolded, were raised in mice. These antibodies were probed for binding to native, immobilized and receptor-bound IgE, respectively, providing tools for the identification of the indicated stable epitopes. RESULTS: Two of the generated antibodies (8E7, 3G9) discriminate between IgE in solution and IgE attached to FcepsilonRIalpha, pointing towards a steric rearrangement within Cepsilon3 induced upon receptor binding. The described antibodies represent tools for studying the mechanism of the Fcepsilon-FcepsilonRIalpha interaction and may be of diagnostic value since serum IgE from various human donors was differently recognized by 8E7, which is indicative for naturally occurring IgE molecules with different steric conformation. CONCLUSION: The presented data support the hypothesis of a conformational change within IgE Cepsilon3 upon receptor binding by showing that monoclonal antibodies raised against recombinant Cepsilon3 differently recognize soluble and receptor-bound IgE. The presence of an IgE portion in sera of human donors that is recognized by 8E7 indicates the existence of IgE molecules in different steric conformations in human blood, which may be related to pathologic parameters.

Antibodies, Monoclonal↗
PubMed1999

Antibody production without animals.

Research always depended on animals or directly on human beings for the generation of antibodies with new specificities. With the opportunity to clone the human antibody repertoire, it is now also feasible to tap the human immune system and to search for new antibody specificities in antibody gene libraries [1, 2]. Phage display technology has fostered these approaches as it allows even rare specificities to be identified in libraries of a complexity of 10(8) to 10(10) [3, 4]. While most of the work in this field is still based on immune libraries, that is, B-cells derived from immunised animals or human individuals, more recent approaches allow even the necessity for immunisation to be by-passed. Synthetic antibody libraries exist of a complexity comparable to the natural immune system of mammals, so that theoretically every imaginable antibody may be detected and then used either for diagnostic or therapeutic purposes [5-7]. Thus, these new gene technology approaches make it possible to generate antibodies completely independently of animals. It may be that animals will only be used in the future to study the immune system of the animal.

Animal Welfare↗
PubMed1998

[Hepatocellular carcinoma: interdisciplinary treatment concept].

In most cases hepatocellular carcinoma develops in a cirrhotic liver. The stage of the cirrhosis and the number and size of the liver tumors are decisive for the prognosis and the individual treatment possibilities. For each therapeutic procedure there are specific contraindications which have to be considered. In a prospective comparative study of different established methods of treatment, i.e. hepatic resection, percutaneous ethanol instillation, transcatheter arterial embolisation and tamoxifen therapy, tumor response, survival time, complications and factors, determining prognosis are analysed. The therapeutic concept of the study can thereby be used as a model for the interdisciplinary treatment of hepatocellular carcinoma.

Carcinoma, Hepatocellular↗