Systemic immunological changes induced by administration of grass pollen allergens via the oral mucosa during sublingual immunotherapy.

Twenty-four patients suffering from grass pollen allergy underwent sublingual immunotherapy (SLIT) with standardized grass pollen extract for 1 year. In order to investigate immunological changes induced by the administration of allergens via the oral mucosa, the SLIT-spit method was applied. The cumulative dose of approximately 80 microg of major allergen (grass group 5 allergen), was relatively low. During the time of treatment, we could observe a significant increase in the levels of specific IgG and IgG4 antibodies. However, the titers of allergen-specific IgE antibodies showed a significant increase in the course of SLIT as well. Analyzing lymphoproliferative responses, a significant decrease in reactivity in response to stimulation with complete grass pollen extract (p = 0. 001) and to recombinant Phl p 1 (a major allergen of timothy grass, p<0.001) could be observed, indicating the induction of immunological tolerance. Proliferative responses to a control antigen (tetanus toxoid) were not influenced by the treatment. At different time points during SLIT, allergen (Phl p 1)-specific T cell clones (TCC) were established from the peripheral blood of the patients. Cytokine production by allergen-stimulated T cells did not reveal any changes consistent with immune deviation, i.e. the ratio of Th1/Th2 TCC did not change during SLIT. In conclusion, we provide evidence that sublingual treatment leads to systemic changes in immunoreactivity to the administered allergen.

Expression of complement regulator proteins in primary and metastatic malignant melanoma.

The expression of complement regulatory antigens C3b/C4b receptor, (CD35) membrane cofactor protein (CD46), decay accelerating factor (CD55), and homologous restriction factor 20 (CD59) was determined immunohistochemically on ten primary malignant melanomas, 16 metastatic lesions, and ten melanocytic nevi. All of the melanocytic nevi and 9/10 primary melanomas showed both expression of CD46 and CD59. In one primary melanoma lacking CD46, expression of CD35 could be detected. In metastatic melanoma, 9/16 metastases were CD46+/CD59+, two were CD46-/CD59+, one CD46+/CD59-, and four CD46-/CD59-. Additionally, CD55 could be detected in two CD46+/CD59+ metastases, and CD35 in one. Expression or lack of complement regulatory antigens did not correlate with the expression of GD2, GD3, HMB-45 or S-100. In conclusion, some cases of metastatic melanoma show loss of normal expression of complement regulatory proteins. This might have implications on the immune response or the efficacy of immune therapy in malignant melanoma.

T cell receptor CDR3 sequences and recombinant T cell receptors. Prerequisites for developing ligands to interfere with T cell receptor binding to the MHC/peptide complex.

BACKGROUND: The interaction of T cell receptors (TCRs) with peptide fragments bound to major histocompatibility complex (MHC) molecules is central to the initiation and propagation of most immune responses. In order to understand and control the molecular interactions underlying T cell recognition of MHC/peptide complexes, recent efforts have focused on the production of recombinant soluble forms of the TCR heterodimer. METHODS: TCRA variable (TCRAV) and TCRBV sequences used by human T cell clones were amplified by PCR, cloned and sequenced. The deduced amino acid sequences of the complementarity determining region (CDR) 3 loops of TCRs specific for the same allergenic epitope were compared. V region genes of a selected TCR were expressed as a single-chain (sc) molecule in the periplasm of Escherichia coli. RESULTS: Conserved amino acid motifs specific for allergenic peptides of Bet v 1 and Phl p 1 were identified in CDR3 sequences of TCRs. A recombinant scTCR was produced. The ratio of insoluble to soluble material was 1:1. The recombinant protein was of the correct size and showed no signs of degradation. CONCLUSIONS: Conservation of amino acid motifs in CDR3 loops of TCRs specific for the same allergen fragments indicated that the three-dimensional structure of the CDR3 was determined by the presented peptide. The recombinant scTCR will be used to identify ligands for the CDR3s from random peptide libraries to interfere with TCR binding to the MHC/peptide complex.

Immunological changes during specific immunotherapy of grass pollen allergy: reduced lymphoproliferative responses to allergen and shift from TH2 to TH1 in T-cell clones specific for Phl p 1, a major grass pollen allergen.

BACKGROUND AND OBJECTIVE: The mechanisms operative in specific immunotherapy (SIT) of Type I allergy are not completely understood. In the present study we evaluated immunological changes during SIT in pollinosis. METHOD: Eight patients suffering from pollinosis (monosensitized to grass pollen) were treated with conventional SIT. All subjects had IgE specific for Phl p 1, a major allergen of timothy grass. In vitro changes in the immunological reactivity to grass pollen extract and to recombinant Phl p 1 were evaluated. Subjects were examined at three occasions: before, after 3 months and after 1 year of SIT. RESULTS: Serological analysis revealed a marked increase of grass pollen- and Phl p 1-specific IgG, titres of specific IgE did not change significantly. Lymphoproliferative responses to grass pollen extract and rPhl p 1 were reduced already after 3 months of treatment. Accordingly, the cloning efficiency for Phl p 1-specific T-cell clones (TCC) dropped markedly in all patients. The majority of allergen-specific TCC raised before SIT revealed a TH2-like pattern of cytokine production, TCC established after SIT revealed TH1 characteristics. This shift was due to a decrease in IL-4 rather than an increase in IFN-production by T cells. Investigations of the epitopes recognized by T cells before and after SIT did not reveal the outgrowth of new ('protecting') specificities. We could not observe induction of allergen-specific CD8+ lymphocytes (supressor cells). CONCLUSION: Our data indicate that -- on the level of TH lymphocytes -- SIT induces tolerance to the allergen and a modulation of the cytokine pattern produced in response to allergen stimulation.

Bet v 1, the major birch pollen allergen, conjugated to crystalline bacterial cell surface proteins, expands allergen-specific T cells of the Th1/Th0 phenotype in vitro by induction of IL-12.

Modulation of allergic immune responses by using adequate adjuvants is a promising concept for future immunotherapy of type I hypersensitivity. In the present study, recombinant Bet v 1 (rBet v 1, the major birch pollen allergen) was conjugated to cross-linked crystalline surface layer proteins (SL) derived from Gram-positive eubacteria. T cell lines (TCL) and clones (TCC) were established from peripheral blood of birch pollen-allergic patients. TCL and TCC were induced either using rBet v 1 alone or rBet v 1/SL conjugates (rBet v 1/SL) as initial antigen stimulus. Cytokine production after re-stimulation with rBet v 1 was investigated. TCL initiated with rBet v 1/SL showed significantly increased IFN-gamma production as compared to rBet v 1 -selected TCL. TCC were established from TCL of five patients. As expected, the majority of CD4+ TCC induced by rBet v 1 (55%) displayed a Th2-like pattern of cytokine production. However, only 21% of Bet v 1-specific TCC isolated from TCL established with the Bet v 1/SL revealed this phenotype. The majority of SL-specific TCC (80%) belonged to the Th1 phenotype. In cultures of peripheral blood mononuclear cells, both, SL and Bet v 1/SL (but not rBet v 1) stimulated the production of high levels of IL-12, a pivotal mediator of Th1 responses. Moreover, stimulation of rBet v 1-induced TCC with rBet v 1/SL led to an increased IFN-gamma production. This effect could be reversed by neutralizing anti-IL-12 mAb. Together these results indicate an adjuvant effect of SL mediated by IL-12. Our results indicate that bacterial components, such as SL, displaying adjuvant effects may be suitable for immunotherapeutical vaccines for type I allergy.

Food allergy to honey: pollen or bee products? Characterization of allergenic proteins in honey by means of immunoblotting.

OBJECTIVE: To characterize the allergenic components of honey, 23 patients allergic to honey were investigated. All displayed allergic symptoms after ingestion of honey or honey-containing products, ranging from itching in the oral mucosa to severe systemic symptoms to anaphylactic shock. METHODS AND RESULTS: Immunoblot analyses of the patients' sera revealed IgE binding to proteins at a molecular mass of 54 kd, 60 kd, 72 kd, or to a 30 kd/33 kd double band, or to both in sunflower honey extracts. The three bands corresponding to higher molecular mass proteins could also be detected in the three other kinds of honey (locust tree, European chestnut and forest honey) that were tested and represented bee products because IgE binding to these proteins was inhibited by extracts of honeybee heads and extracts of isolated bee venom sacs. The 30 kd/33 kd bands could be identified as sunflower honey-specific. When testing sera from patients allergic to bee venom with honey extracts, in seven of 10 cases IgE binding to bee-specific components could be observed. CONCLUSION: Both proteins derived from secretions of pharyngeal and salivary glands of honeybee heads and pollen proteins contained in the honey cause allergic reactions to honey.

Characterization of allergen (Bet v 1)-specific T cell lines and clones from non-allergic individuals.

The immune response towards allergens in non-allergic healthy individuals was investigated. T cell lines (TCL) with specificity for Bet v 1, the major birch pollen allergen, were established and analysed for epitope specificity. 49 T cell clones (TCC) specific for Bet v 1 were isolated from TCLs. All TCCs revealed the Th phenotype. Cytokine production in response to specific stimulation revealed a majority of Th clones producing interleukin (IL)-4 and interferon (IFN)-gamma; however, most TCCs revealed a low IL-4/IFN-gamma ratio. Immunoblot revealed Bet v 1-specific IgG in non-allergic individuals whereas no IgE could be detected. Our results indicate that T cells from allergic and non-allergic individuals recognize the same epitopes on allergenic molecules, leading to activation, which then results in a differential production of cytokines and consequently to differential isotype switching in allergen-specific B cells.

Nonallergic individuals recognize the same T cell epitopes of Bet v 1, the major birch pollen allergen, as atopic patients.

The immune response toward allergens in nonallergic healthy individuals was investigated. To boost immune responses, two injections of birch pollen extract were administered to five nonallergic volunteers. T cell lines (TCL) with specificity for Bet v 1, the major birch pollen allergen, were established and analyzed for epitope specificity using overlapping peptides. Forty-nine T cell clones (TCC) specific for Bet v 1 were isolated from TCLs. Comparison with TCL and TCC established from birch pollen-allergic patients was performed. All TCC revealed the Th phenotype. Epitope specificities of TCL and TCC from nonatopics were identical to those found in allergic individuals. No association between MHC class II molecules and particular epitopes could be observed. In nonallergic as well as in allergic individuals, cytokine production in response to specific stimulation revealed a majority of Th-clones producing IL-4 and IFN-gamma. However, TCC derived from atopic individuals revealed a higher IL-4/IFN-gamma ratio. Immunoblot and ELISA revealed Bet v 1-specific IgG in nonallergic individuals before and after booster injections, but no IgE could be detected. High levels of Bet v 1-specific IgG and IgE could be detected in birch pollen-allergic patients. It can be concluded, that nonatopic and allergic individuals display the same repertoire of T cell specificities. In allergic individuals, the activation of allergen-specific TCC leads to a higher ratio of produced IL-4 vs IFN-gamma, which is responsible for enhanced IgE production.

Prevention of peritoneal adhesions in the rat with sustained intraperitoneal dexamethasone delivered by a novel therapeutic system.

The rat caecal crush model for the production of intraperitoneal adhesions was optimized with respect to an objective adhesion grading system based on the determination of the incidence of adhesions and the caecal adherent surface area. Intraperitoneal saline solution given before closure of the peritoneum significantly reduced the severity but not the incidence of adhesions. The addition of dexamethasone crystal suspension led to somewhat lower incidence of adhesions but the mean adherent surface area values were not different from the saline regimen. However, sustained release of dexamethasone by use of intraperitoneal biodegradable poly(lactide-co-glycolide) microparticles as a novel therapeutic system further reduced both the incidence and the severity of adhesions. Despite of the differences in therapeutic effects glucocorticoid-associated local and systemic adverse effects were similar in both regimens. As effective adhesion prophylaxis with glucocorticoids appears to be a balance between benefit and adverse reactions, optimal drug delivery is mandatory. The novel biodegradable therapeutic system is superior to the intraperitoneal crystal suspension regimen in the rat and should be tested further in clinical trials.