Previously, defined naturally occurring isoforms of allergenic proteins were classified as hypoallergens and therefore suggested as an agent for immunotherapy in the future. In this paper, we report for the first time the molecular background of hypoallergenicity by comparing the immunological behavior of hyperallergenic Betula verrucosa major Ag 1a (Bet v 1a) and hypoallergenic Bet v 1d, two isoforms of the major birch pollen allergen Betula verrucosa 1. Despite their cross-reactivity, Bet v 1a and Bet v 1d differ in their capacity to induce protective Ab responses in BALB/c mice. Both isoforms induced similar specific IgE levels, but only Bet v 1d expressed relevant titers of serum IgGs and IgAs. Interestingly, hypoallergenic Bet v 1d activated dendritic cells more efficiently, followed by the production of increased amounts of Th1- as well as Th2-type cytokines. Surprisingly, compared with Bet v 1a, Bet v 1d-immunized mice showed a decreased proliferation of regulatory T cells. Crystallographic studies and dynamic light scattering revealed that Bet v 1d demonstrated a high tendency to form disulfide-linked aggregates due to a serine to cysteine exchange at residue 113. We conclude that aggregation of Bet v 1d triggers the establishment of a protective Ab titer and supports a rationale for Bet v 1d being a promising candidate for specific immunotherapy of birch pollen allergy.

Staphylococcus aureus is a human pathogen of growing clinical significance, owing to its increasing levels of resistance to most antibiotics. Infections range from mild wound infections to severe infections such as endocarditis, osteomyelitis and septic shock. Adherence of S. aureus to human host cells is an important step, leading to colonization and infection. Adherence is mediated by a multiplicity of proteins expressed on the bacterial surface, including clumping factor B. In this study, we aimed to identify new targets of clumping factor B in human keratinocytes by undertaking a genome-wide yeast two-hybrid screen of a human keratinocyte cDNA library. We show that clumping factor B is capable of binding cytokeratin 8 (CK8), a type II cytokeratin. Using a domain-mapping strategy we identified amino acids 437-464 as necessary for this interaction. Recombinantly expressed fragments of both proteins were used in pull-down experiments and confirmed the yeast two-hybrid studies. Analysis with S. aureus strain Newman deficient in clumping factor B showed the clumping factor B-dependence of the interaction with CK8. We postulate that the clumping factor B-CK8 interaction is a novel factor in S. aureus infections.

The switch of B cells expressing membrane-bound Igs, which serve as antigen receptors, to antibody-secreting plasmablasts and finally to non-dividing, long-lived plasma cells (PCs) lacking an antigen receptor, marks the terminal differentiation of a B cell. Antibody-secreting PCs represent the key cell type for the maintenance of a proactive humoral immunological memory. Although some populations of long-lived PCs persist in the spleen, most of them return to their 'place of birth' and travel to the bone marrow or invade inflamed tissues, where they survive up to several months in survival niches as resident, immobile cells. Existing data strongly support the notion that isotype-specific receptor signalling influences the migration behaviour of plasmablasts to the bone marrow. The recent observation in the murine system that the immigration of plasmablasts and the final differentiation to long-lived PCs in the bone marrow is dependent on the expressed B-cell isotype and the related expression of chemokine receptors leads to the conclusion that during a T-helper type 2 (Th2)-mediated immune response in wild type mice, IgE plasmablasts do not have the same chance to contribute to long-lived PC memory as IgG1 plasmablasts. The overall limited humoral IgE memory additionally restricts the quantity of IgE Igs in the serum.

Epidemiological studies have suggested inverse associations between allergic diseases and malignancies. As a proof of concept for the capability of immunoglobulin E (IgE) to destruct tumor cells, several experimental strategies have evolved to specifically target this antibody class towards relevant tumor antigens. It could be demonstrated that IgE antibodies specific to overexpressed tumor antigens have been superior to any other immunoglobulin class with respect to antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) reactions. In an alternative approach, IgE nonspecifically attached to tumor cells proved to be a powerful adjuvant establishing tumor-specific immune memory. Active Th2 immunity could also be achieved by applying an oral immunization regimen using mimotopes, i.e. epitope mimics of tumor antigens. The induced IgE antibodies could be cross-linked by live tumor cells leading to tumoricidic mediator release. Thus, IgE antibodies may not only act in natural tumor surveillance, but could possibly also be exploited for tumor control in active and passive immunotherapy settings. Thereby, eosinophils, mast cells and macrophages can be armed with the cytophilic IgE and become potent anti-tumor effectors, able to trace viable tumor cells in the tissues. It is strongly suggested that the evolving new field AllergoOncology will give new insights into the role of IgE-mediated allergy in malignancies, possibly opening new avenues for tumor therapy.

Background: The position of the tonsils which belong to the mucosa-associated lymphoid tissues (MALT), implies a key role as secondary lymphoid organ in initiating immune responses against various antigens. While the IgE antibody-mediated release of mediators constitutes the immunopathological basis for immediate symptoms, much less is known concerning the role of allergen-specific responses of isotypes other than IgE. In order to investigate the impact of an allergen-specific immune reaction on the local immune response in palatine tonsils, the expression of IgA + plasma cells and the distribution of proliferating cells in Bet v 1-(Betula verrucosa 1) allergic and non-allergic patients were detected. Methods: Using immunohistological methods, IgA + plasma cells and K167 + cells were stained in cryosections of tonsils from 20 patients according to chronic tonsillitis (Bet v 1-allergic/non-allergic patients, n = 10). The distribution of IgA + plasma- and Ki67 + -proliferating cells was analyzed. Results: Our analyses showed a differential distribution and a significantly (p < 0.01) diminished expression of IgA + plasma cells in tonsils of Bet v 1-allergic patients compared to non-allergic patients. Further, tonsils of allergic and non-allergic patients did not show differences in the distribution of proliferating cells. However, tonsils of both investigated groups were comparable concerning their number and size of secondary lymphoid follicles and their morphologic structures. Conclusions: Altogether, these data may lead to the assumption that the reduction of IgA + plasma cells within the epithelial microenvironment of the crypts and the extrafollicular areas in allergic individuals results in a reduced ability to mount a protective immune response against allergens. The comparable number and size of follicles and the distribution of proliferating cells in the tonsillar germinal centers may reflect the ongoing germinal center reaction.

Signalling through the B cell antigen receptor (BCR) is required for peripheral B lymphocyte maturation, maintenance, activation and silencing. In mature B cells, the antigen receptor normally consists of two isotypes: membrane IgM and IgD (mIgM, mIgD). Although the signals initiated from both isotypes differ in kinetics and intensity, in vivo, the BCR of either isotype seems to be able to compensate for the loss of the other, reflected by the mild phenotypes of mice deficient for mIgM or mIgD. Thus, it is still unclear why mature B cells need expression of mIgD in addition to mIgM. In the present paper, we used the B cell line Bcl1 and investigated the isotype-specific antigen internalization in dependence of co-stimulation of the reciprocal isotype and analysed whether the signal initiated from mIgM is modulated through signalling from mIgD and vice versa. We clearly showed that cross-linkage of mIgM decreases the rate of mIgD mediated antigen internalization and interpret this influence as a unilateral mIgM mediated control on signals initiated at mIgD.

Background: The position of the tonsils which belong to the mucosa-associated lymphoid tissues (MALT), implies a key role as secondary lymphoid organ in initiating immune responses against various antigens. While the IgE antibody-mediated release of mediators constitutes the immunopathological basis for immediate symptoms, much less is known concerning the role of allergen-specific responses of isotypes other than IgE. In order to investigate the impact of an allergen-specific immune reaction on the local immune response in palatine tonsils, the expression of IgA(+) plasma cells and the distribution of proliferating cells in Bet v 1(Betula verrucosa 1) allergic and non-allergic patients were detected. Methods: Using immunohistological methods, IgA(+) plasma cells and Ki67(+) cells were stained in cryosections of tonsils from 20 patients according to chronic tonsillitis (Bet v 1-allergic/non-allergic patients, n = 10). The distribution of IgA(+) plasma- and Ki67(+)-proliferating cells was analyzed. Results: Our analyses showed a differential distribution and a significantly (p < 0.01) diminished expression of IgA(+). plasma cells in tonsils of Bet v 1-allergic patients compared to non-allergic patients. Further, tonsils of allergic and non-allergic patients did not show differences in the distribution of proliferating cells. However, tonsils of both investigated groups were comparable concerning their number and size of secondary lymphoid follicles and their morphologic structures. Conclusions: Altogether, these data may lead to the assumption that the reduction of IgA+ plasma cells within the epithelial microenvironment of the crypts and the extrafollicular areas in allergic individuals results in a reduced ability to mount a protective immune response against allergens. The comparable number and size of follicles and the distribution of proliferating cells in the tonsillar germinal centers may reflect the ongoing germinal center reaction.

Immunoglobulin E (IgE) was the last of the immunoglobulins discovered. It is present in very low amounts (nano- to micro-gram per ml range) in the serum of normal healthy individuals and normal laboratory mouse strains and has a very short half-life. This contrasts with the other immunoglobulin classes, which are present in much higher concentrations (micro- to milligram per ml range) and form a substantial component of serum proteins. Immunoglobulins play a role in homeostatic mechanisms and they represent the humoral arm of defence against pathogenic organisms. Since IgE antibodies play a key role in allergic disorders, a number of approaches to inhibit IgE antibody production are currently being explored. In the recent past the use of nonanaphylactic, humanized anti-IgE antibodies became a new therapeutic strategy for allergic diseases. The therapeutic rational beyond the idea derives from the ability of the anti-IgE antibodies to bind to the same domains on the IgE molecule that interact with the high-affinity IgE receptor, thereby interfering with the binding of IgE to this receptor without cross-linking the IgE on the receptor (nonanaphylactic anti-IgE antibodies). Treatment with anti-IgE antibodies leads primarily to a decrease in serum IgE levels. As a consequence thereof, the number of high-affinity IgE receptors on mast cells and basophils decreases, leading to a lower excitability of the effector cells reducing the release of inflammatory mediator such as histamine, prostaglandins and leukotrienes. Experimental studies in mice indicate that injection of some monoclonal anti-IgE antibodies also inhibited IgE production in vivo. The biological mechanism behind this reduction remains speculative. A possible explanation may be that these antibodies can also interact with membrane bound IgE on B cells, which could interfere the IgE production.

RATIONALE: Clinical manifestations of type 1 hyperreactivity are triggered when allergens cross-link effector cell-bound IgE and aggregate the underlying high affinity FcεRI.While the IgE antibody-mediated release of mediators constitutes the immunopathological basis for immediate symptoms, much less is known concerning the role of allergen-specific responses of isotypes other than IgE.METHODS: Our analysis of the major birch pollen (Betv1) specific immune response showed for the first time that Betv1 specific memory B cells are present in tonsils and peripheral blood of both atopic and nonatopic individuals.RESULTS: RT-PCR of single Betv1 specific B cells revealed highly mutated sequences mainly of the IgG and IgA isotype.Striking was the high percentage of VH3-30 or VL3l gene rearrangements.This result was confirmed by an in vitro antibody affinity selection via phage panning of a human VH/VL phage library (Griffin.1)using recombinant Betv1.Recombinant antibodies with VH3-30 or VL3l rearrangement had the highest relative affinities as measured by plasmon resonance technology.CONCLUSIONS: As Betv1 plasma cells were detectable in tonsils only in atopic individuals, the question rises, how the differentiation of Betv1 specific memory B cells in non-atopic individuals is controlled and why Betv1 specific IgE antibodies as in case of atopic disease are not synthesized.

Ziel der Studie war die Charakterisierung und der Vergleich der Betv1 (Betula verrucosa) spezifischen Immunantwort von Birkenpollen-Allergikern und Gesunden in Bezug auf die Lokalisation Betv1 spezifischer B-Zellen in Tonsillen und die Detektion im peripheren Blut. Auf molekularer Ebene wurde das Sequenz Repertoire der Allergen-spezifischen Antikörper (AK) untersucht.Material und Methoden Hierfür wurden Tonsillen/ Serum von je 30 Allergikern/ Gesunden untersucht. Die Tonsillen wurden histologisch auf Betv1-spezifische B-Zellen, das Serum auf spezifische Betv1-AK untersucht. Zusätzlich wurden nach Dichtegradienten-Zentrifugation mononukleäre Zellen aus dem Blut isoliert und mit durchflusszytometrischen Methoden (FACS) analysiert. Aus den Einzelzellen wurden mit RT-PCR die schweren und leichten AK- Ketten der spezifischen B Zellen amplifiziert und sequenziert.Ergebnisse Die Untersuchung der Tonsillen ergab, dass die Anzahl der IgE- Plasmazellen bei Allergikern signifikant erhöht war. Erstmals konnten Betv1-spezifische Plasmazellen bei Allergikern nachgewiesen werden. Im Serum wurde in beiden Kohorten Betv1-spezifisches IgG, nur im Serum der Allergiker Betv1-spezifisches IgE bestimmt. Auf Einzelzellebene waren Allergen-spezifische Zellen in beiden Gruppen nachweisbar, bei Allergikern war ihre Anzahl signifikant erhöht.Schlussfolgerung Neben Birkenpollen-spezifischen IgG und IgE AK bei Allergikern produzieren auch Gesunde diese AK vom IgG Isotyp. Beide entwickeln einen spezifischen Gedächtnispool, was für einen zusätzlichen Kontrollmechanismus bei Gesunden spricht.

Rationale Striking IgE production at the root with generating antibodies against the extracellular portion of the transmembrane domain (EMPD) of IgE could be a further strategy concerning the therapy of allergic diseases. Methods The phage display biopanning procedure was used for selection of single chain fragments (scFv) recognizing the Extra Membrane Proximal Domain (EMPD). Results So far, we were able to isolate two specific single chain antibodies. Showing specificity in Elisa- as well as Biacore-analysis for the EMPD-region, the sequence of these single chains will be the basis for the generation of Fab-, F(ab')2-fragments and whole antibodies, by cloning procedures. Expressed recombinant antibodies and DNA-vaccine-constructions will be used for mice immunization experiments. Conclusions The advantage of anti-mIgE antibodies would be the inhibition of IgE secretion before sIgE production starts. Our hypothesis suggests that cross linking mIgE receptors by anti-EMPD antibodies without the appropriate T cell help leads to direct clonal deletion and/or clonal anergy of the total mIgE B cell population.

Rationale The expression of IgE is tightly regulated on different levels. Preliminary data from our laboratories suggest a regulation based on the expression of the membrane mound IgE. Methods A step forward in the analysis of this tight regulation was done with the construction of knock-in mice with composite and/or chimeric poly(A) sites influencing the post-transcriptional regulation. Results Expression of mRNA for the secreted form of IgE is favored over that for the membrane form in resting B cells. The ratio between the amount of secreted vs. the amount of membrane-bound Ig that is produced by a single cell is determined by the efficiency with which the internal or external polyadenylation sites are used and by the stability of the ensuing mRNA's. Conclusions Factors that influence the alternative polyadenylation are largely unknown. However, because expression of mIgE is essential for recruitment of IgE-producing cells in the immune response, clarification of this issue is of great importance. So far, IgE regulation is evident on the level of DNA recombination (switch), transcription and RNA processing. It is not inconceivably that also post-translational and post-transcriptional processes may influence the expression of membrane-bound IgE.