Blocking antibodies induced by specific allergy vaccination prevent the activation of CD4+ T cells by inhibiting serum-IgE-facilitated allergen presentation.
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Allergen-specific CD4+ T lymphocytes are activated at extremely low allergen concentrations in vivo as a result of serum-facilitated allergen presentation (S-FAP). It is not clear at present if specific allergy vaccination (SAV) has an effect on this mechanism. Here we show that birch allergen-specific serum-IgE facilitates the presentation of Bet v 1, the major birch pollen allergen, to Bet v 1-specific CD4+ T lymphocytes by a factor of >100. This process is CD23 mediated, could be detected in sera from the majority of birch-allergic patients, and was clearly dose dependent. S-FAP of Bet v 1 was inhibited in patients undergoing long-term birch SAV, but not by sera from patients undergoing grass SAV, indicating that birch-specific Abs are involved. This resulted in decreased proliferation and IL-4, IL-5, IL-10, and IFN-gamma production of Bet v 1-specific T cells. The inhibition was already noted after 3-9 mo of SAV and could not be solely explained by increased serum levels of birch-specific IgG4. When IgG- and IgA/IgM-containing fractions of long-term SAV sera were used to inhibit S-FAP, only IgG-containing fractions were shown to inhibit S-FAP. These results indicate that blocking IgG Abs induced by SAV inhibits the occurrence of S-FAP at very low allergen concentrations, resulting in significantly higher allergen threshold levels to obtain T cell proliferation and cytokine production and thus allergen-induced late-phase responses. (+info)
Allergen mimotopes in food enhance type I allergic reactions in mice.
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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. (+info)
TCR-mediated activation of allergen-specific CD45RO(+) memory T lymphocytes results in down-regulation of cell-surface CXCR4 expression and a strongly reduced capacity to migrate in response to stromal cell-derived factor-1.
(19/684)
The selective migration of functional T(h) lymphocyte subsets with different cytokine production profiles into inflamed tissue is likely to depend on the state of activation of the cells, as well as on the differential expression of various adhesion molecules and chemokine receptors. In this study, we have analyzed the effect of allergen-specific activation on the expression of the chemokine receptor CXCR4 on T lymphocytes. We show that stimulation of peripheral blood mononuclear cells from atopic patients with the allergen Der p results in down-regulation of CXCR4 surface expression on Der p-activated CD25(+)CD45RO(+) antigen-specific memory cells which was caused by a decrease in CXCR4 gene transcription and did not seem to be mediated by endogenous cytokines, such as IFN-gamma. In contrast, however, CXCR4 surface expression was enhanced on naive CD25(-)CD45RO(-) and resting CD25(-)CD45RO(+) memory T cells, as a result of the presence of endogenous IL-4, most likely produced by Der p-activated memory T cells. Antigen-specific CD25(+)CD45RO(+) T lymphocytes, purified 7 days after stimulation with Der p, had a strongly reduced capacity to migrate in response to stimulation with stromal cell-derived factor (SDF)-1, the ligand for CXCR4. Together, these results suggest that differential expression of CXCR4 on activated and resting T cells is due to the counteracting effects of TCR-mediated down-regulation and IL-4-mediated up-regulation of this chemokine receptor respectively, and furthermore indicate that antigen-activated memory T cells are unlikely to migrate into inflamed tissue in response to SDF-1. (+info)
Direct kinetic evidence for folding via a highly compact, misfolded state.
(20/684)
The 2 S seed storage protein, sunflower albumin 8 (SFA-8), contains an unusually high proportion of hydrophobic residues including 16 methionines (some of which may form a surface hydrophobic patch) in a disulfide cross-linked, alpha-helical structure. Circular dichroism and fluorescence spectroscopy show that SFA-8 is highly stable to denaturation by heating or chaotropic agents, the latter resulting in a reversible two-state unfolding transition. The small m(U) (-4.7 M(-1) at 10 degrees C) and DeltaC(p) (-0.95 kcal mol(-1) K(-1)) values indicate that relatively little nonpolar surface of the protein is exposed during unfolding. Commensurate with the unusual distribution of hydrophobic residues, stopped-flow fluorescence data show that the folding pathway of SFA-8 is highly atypical, in that the initial product of the rapid collapse phase of folding is a compact nonnative state (or collection of nonnative states) that must unfold before acquiring the native conformation. The inhibited folding reaction of SFA-8, in which the misfolded state (m(M) = -0.95 M(-1) at 10 degrees C) is more compact than the transition state for folding (m(T) = -2.5 M(-1) at 10 degrees C), provides direct kinetic evidence for the transient misfolding of a protein. (+info)
Mechanistic basis for coding end sequence effects in the initiation of V(D)J recombination.
(21/684)
V(D)J recombination is directed by recombination signal sequences. However, the flanking coding end sequence can markedly affect the frequency of the initiation of V(D)J recombination in vivo. Here we demonstrate that the coding end sequence effect can be qualitatively and quantitatively recapitulated in vitro with purified RAG proteins. We find that coding end sequence specifically affects the nicking step, which is the first biochemical step in RAG-mediated cleavage. The subsequent hairpin formation step is not affected by the coding end sequence. Furthermore, the coding end sequence effect can be ablated by prenicking the substrate, indicating that the coding end effect is specific to the nicking step. In reactions in which both 12- and 23-substrates are present, a suboptimal coding end sequence on one signal can slow down hairpin formation at the partner signal, a result consistent with models in which coordination between the signals occurs at the hairpin formation step. The coding end sequence effect on nicking and the coupling of the 12- and 23-substrates explains how hairpin formation can be rate limiting for some 12/23 pairs, whereas nicking can be rate limiting when low-efficiency coding end sequences are involved. (+info)
Component-resolved diagnosis (CRD) of type I allergy with recombinant grass and tree pollen allergens by skin testing.
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The diagnosis of Type I allergy is based on the measurement of allergen-specific IgE antibodies and on provocation with allergens, most frequently conducted by skin testing. Both forms of diagnosis are currently performed with allergen extracts that are difficult to standardize regarding their allergen contents, and which contain additional undefined nonallergenic components. We report the expression in Escherichia coli and purification of some of the most relevant timothy grass- and birch pollen allergens. Recombinant timothy grass- (rPhl p 1, rPhl p 2, rPhl p 5) and birch pollen (rBet v 1, rBet v 2) allergens were purified and used for the measurement of allergen-specific IgE and IgG subclass responses as well as for skin prick testing in 55 pollen allergic patients and 10 nonatopic individuals. Results obtained showed that the recombinant allergens allowed in vivo allergy diagnosis in 52 of 54 of the grass pollen and in 35 of 36 of the birch pollen allergic patients. Positive skin reactions were observed almost exclusively in patients containing detectable allergen-specific IgE antibodies but not in the nonatopic group; however, sensitivity to a given allergen as measured by skin reactivity was weakly correlated with the levels of allergen-specific IgE. Our results demonstrate that recombinant allergens can be used for component-resolved skin test diagnosis (CRD) of the patients' allergen sensitization profile, whereas allergen extracts at best allow to identify allergen-containing sources. CRD may thus represent the basis for novel forms of patient-tailored immunotherapy. (+info)
Inclusion of a furin-sensitive spacer enhances the cytotoxicity of ribotoxin restrictocin containing recombinant single-chain immunotoxins.
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Chimaeric toxins have considerable therapeutic potential to treat various malignancies. We have previously used the fungal ribonucleolytic toxin restrictocin to make chimaeric toxins in which the ligand was fused at either the N-terminus or the C-terminus of the toxin. Chimaeric toxins containing ligand at the C-terminus of restrictocin were shown to be more active than those having ligand at the N-terminus of the toxin. Here we describe the further engineering of restrictocin-based chimaeric toxins, anti-TFR(scFv)-restrictocin and restrictocin-anti-TFR(scFv), containing restrictocin and a single chain fragment variable (scFv) of a monoclonal antibody directed at the human transferrin receptor (TFR), to enhance their cell-killing activity. To promote the independent folding of the two proteins in the chimaeric toxin, a linear flexible peptide, Gly-Gly-Gly-Gly-Ser, was inserted between the toxin and the ligand to generate restrictocin-linker-anti-TFR(scFv) and anti-TFR(scFv)-linker-restrictocin. A 12-residue spacer, Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu, containing the recognition site for the protease furin, was incorporated between the toxin and the ligand to generate restrictocin-spacer-anti-TFR(scFv) and anti-TFR(scFv)-spacer-restrictocin. The incorporation of the proteolytically cleavable spacer enhanced the cell-killing activity of both constructs by 2-30-fold depending on the target cell line. However, the introduction of linker improved the cytotoxic activity only for anti-TFR(scFv)-linker-restrictocin. The proteolytically cleavable spacer-containing chimaeric toxins had similar cytotoxic activities irrespective of the location of the ligand on the toxin and they were found to release the restrictocin fragment efficiently on proteolysis in vitro. (+info)
Molecular characterization of human IgG monoclonal antibodies specific for the major birch pollen allergen Bet v 1. Anti-allergen IgG can enhance the anaphylactic reaction.
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We report the molecular characterization of five human monoclonal antibodies, BAB1-5 (BAB1: IgG(1); BAB4: IgG(2); BAB2, 3, 5: IgG(4)), with specificity for the major birch pollen allergen, Bet v 1. BAB1-5 were obtained after immunotherapy and contained a high degree of somatic mutations indicative of an antigen-driven affinity maturation process. While BAB1 inhibited the binding of patients IgE to Bet v 1, BAB2 increased IgE recognition of Bet v 1, and, even as Escherichia coli-expressed Fab, augmented Bet v 1-induced immediate type skin reactions. The demonstration that IgG antibodies can enhance allergen-induced allergic reactions is likely to explain the unpredictability of specific immunotherapy. (+info)