Use of a genetic cholera toxin B subunit/allergen fusion molecule as mucosal delivery system with immunosuppressive activity against Th2 immune responses.
ABSTRACT Induction of peripheral tolerance can be facilitated when the antigen is linked to the B subunit of cholera toxin (CTB), an efficient mucosal carrier. In the present study, a genetic fusion molecule of Bet v 1 and CTB was produced to test whether mucosal application of this construct would lead to suppression of Th2 responses. Intranasal pretreatment of BALB/c mice with rCTB-Bet v 1 prior to allergic sensitisation with the allergen significantly decreased IgE but markedly increased allergen-specific IgG2a levels in sera as well as IFN-gamma production of splenocytes. This Th1 shift was supported by an increased T-bet/GATA3 mRNA ratio. IL-5 production within the airways was suppressed after the pretreatment with rCTB-Bet v 1, while local allergen-specific IgA antibodies were markedly enhanced by pretreatment with the construct. Upregulation of Foxp3, IL-10 and TGF-beta mRNA expression was detected in splenocytes after pretreatment with unconjugated allergen but not with the fusion molecule, indicating that antigen conjugation to a mucosal carrier modifies the immunomodulating properties of an antigen/allergen.
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ABSTRACT: Oxidized phospholipids are thought to play a role in the development of atherosclerosis and other chronic inflammatory processes. In this study, we analyzed the expression of inflammatory genes induced by oxidized L-alpha-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholin (OxPAPC) in vitro and in vivo using quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Cultured human umbilical vein endothelial cells (HUVEC) and monocyte-like U937 cells were treated with OxPAPC or lipopolysaccharide (LPS) for 3 h. For in vivo studies, OxPAPC or LPS was injected intravenously into female C57Bl/6J mice and different tissues were isolated after 3 h. We found that both OxPAPC and LPS induced expression of early growth response factor 1 (EGR-1) and monocyte chemoattractant protein 1 (MCP-1) in HUVEC and of JE, the mouse homologue of MCP-1, in liver and heart. Interestingly, OxPAPC but not LPS increased expression of heme oxygenase 1 (HO-1) in U937 cells, HUVEC, aorta, heart, liver, and isolated blood cells. In contrast, E-selectin was selectively induced by LPS, but not by OxPAPC. Finally, OxPAPC-induced expression of HO-1 was blocked by a platelet-activating factor (PAF) receptor antagonist. We conclude that oxidized phospholipids are biologically active in vivo and exert a specific response inducing a pattern of genes that is different from that induced by LPS. In addition, we demonstrate that the quantitative real-time RT-PCR technology is a proper tool to investigate differential inflammatory gene induction in vivo.Vascular Pharmacology 05/2002; 38(4):219-27. · 3.21 Impact Factor
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ABSTRACT: Recent reports have demonstrated that feeding small amounts of antigen conjugated to the B subunit of cholera toxin (CTB) suppress immune responses in experimental models of certain T h 1-based autoimmune diseases. We have established a model of aerosol sensitization leading to T h 2-mediated allergic immune responses in BALB/c mice. In the present study two different antigens, the dietary antigen ovalbumin (OVA) and the inhalant allergen Bet v 1 (the major birch pollen allergen), chemically coupled to recombinant CTB were tested for their potential to influence T h 2-like immune responses. Intranasal administration of OVA–CTB prior to sensitization with OVA led to a significant decrease of antigen-specific IgE antibody levels, but a marked increase of OVA-specific IgG2a antibodies as compared to non-pretreated, sensitized animals. Antigen-specific lympho-proliferative responses in vitro were reduced by 65% in the pretreated group; IL-5 and IL-4, but not IFN-γ, production were markedly decreased in responder cells of lungs and spleens of nasally pretreated mice. In contrast, mucosal administration of rBet v 1–CTB conjugates prior to sensitization led to an up-regulation of allergen-specific IgE, IgG1 and IgG2a, increased in vitro lympho-proliferative responses as well as augmented production of IL-5, IL-4, IL-10 and IFN-γ. Intranasal administration prior to sensitization of unconjugated allergens showed also contrasting effects: OVA could not significantly influence antigen-specific antibody or cytokine production, whereas intranasal pretreatment with unconjugated Bet v 1 suppressed allergen-specific immune responses in vivo and in vitro . These results demonstrated that the two antigens—in conjugated as in unconjugated form—had different effects on the T h 2 immune responses. We therefore conclude that the tolerogenic or immunogenic properties of CTB—and probably also other antigen-delivery systems—strongly depend on the nature of the coupled antigen–allergen.International Immunology 11/1999; · 3.14 Impact Factor
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ABSTRACT: Cholera toxin B subunit (CTB) is an efficient mucosal carrier molecule for the generation of mucosal antibody responses and/or induction of systemic T-cell tolerance to linked antigens. CTB binds with high affinity to GM1 ganglioside cell surface receptors. In this study, we evaluated how conjugation of a peptide or protein antigen to CTB by chemical coupling or genetic fusion influences the T-cell-activating capacity of different antigen-presenting cell (APC) subsets. Using an in vitro system in which antigen-pulsed APCs were incubated with antigen-specific, T-cell receptor-transgenic T cells, we found that the dose of antigen required for T-cell activation could be decreased >10,000-fold using CTB-conjugated compared to free antigen. In contrast, no beneficial effects were observed when CTB was simply admixed with antigen. CTB conjugation enhanced the antigen-presenting capacity not only of dendritic cells and B cells but also of macrophages, which expressed low levels of cell surface major histocompatibility complex (MHC) class II and were normally poor activators of naive T cells. Enhanced antigen-presenting activity by CTB-linked antigen resulted in both increased T-cell proliferation and increased interleukin-12 and gamma interferon secretion and was associated with up-regulation of CD40 and CD86 on the APC surface. These results imply that conjugation to CTB dramatically lowers the threshold concentration of antigen required for immune cell activation and also permits low-MHC II-expressing APCs to prime for a specific immune response.Infection and Immunity 10/2001; · 4.07 Impact Factor