Monomeric and dimeric IgG fractions show differential reactivity against pathogen-derived antigens.

Polyvalent Ig preparations, derived from the pooled plasma of thousands of healthy donors, contain a complex mix of both 'acquired' and natural antibodies directed against pathogens as well as foreign and self/auto antigens (Ag). Depending on their formulation, donor pool size, etc., liquid Ig preparations contain monomeric and dimeric IgG. The dimeric IgG fraction is thought to represent mainly idiotype-antiidiotype Ab pairs. Treatment of all IgG fractions at pH 4 effectively monomerizes the IgG dimers resulting in separated idiotype-antiidiotype Ab pairs and thus in a comparable F(ab')(2) binding site availability of the different IgG fractions. Previously, we identified an increased anti-self-reactivity within the monomerized dimer fraction. This study addressed if, among the different IgG fractions, an analogous preferential reactivity was evident in the response against different pathogen-derived protein and carbohydrate antigens. Therefore, we assessed the activity of total unseparated IgG, the monomeric and dimeric IgG fractions against antigenic structures of bacterial and viral antigens/virulence factors. All fractions showed similar reactivity to protein antigens except for exotoxin A of Pseudomonas aeruginosa, where the dimeric fraction, especially when monomerized, showed a marked increase in reactivity. This suggests that the production of antiidiotypic IgG antibodies contributes to controlling the immune response to certain categories of pathogens. In contrast, the monomeric IgG fractions showed increased reactivity towards pathogen-associated polysaccharides, classically regarded as T-independent antigens. Taken together, the differential reactivity of the IgG fractions seems to indicate a preferential segregation of antibody reactivities according to the nature of the antigen.

Polyphenol-enriched apple extract attenuates food allergy in mice.

BACKGROUND: The immune system may be modulated with nutrition to prevent the development or to treat the symptoms of allergy. Among other foods, consumption of apples has been linked to reduced incidence of atopic dermatitis and respiratory allergy. OBJECTIVE: We evaluated the efficacy and mechanisms of a polyphenol-enriched apple extract in reducing symptoms of food allergy. METHODS: In a model of food allergy to ovalbumin (OVA), BALB/c mice were fed with an apple extract either during sensitization or just before the challenge. After the challenge, allergic symptoms were scored, OVA-specific serum immunoglobulins were determined by ELISA, cytokine production by mesenteric lymph node (MLN) cells was measured by a multiplex assay and gene expression profiles in the intestine were addressed using quantitative real-time PCR. RESULTS: Consumption of the apple extract reduced symptoms of food allergy upon challenge. This was paralleled by reduced levels of intestinal mast cell protease, diminished cytokine secretion by MLN cells and reduced local intestinal mRNA expression of various T-helper type-2 associated and pro-inflammatory genes. Mechanistic studies suggested decrease of mediator release by effector cells and reduction of allergenicity by protein-polyphenol interaction as potential mechanisms responsible for protection. CONCLUSION: Polyphenol-enriched apple extract can attenuate food allergy symptoms in sensitized mice via two distinct possible mechanisms.

Cellular immunity in healthy volunteers treated with an octavalent conjugate Pseudomonas aeruginosa vaccine.

Humoral immunity in response to an octavalent O-polysaccharide-toxin A conjugate Pseudomonas aeruginosa vaccine is well studied, and a phase III clinical study in cystic fibrosis (CF) patients is currently ongoing. In contrast, little is known about cellular immunity induced by this vaccine. Fifteen healthy volunteers were immunized on days 1 and 60. Parameters of cellular immunity were studied before vaccination on day 1, and on day 74. Analyses included flow cytometry of whole blood and antigen-induced proliferation of and cytokine production by lymphocyte cultures. The effects of immunization on the composition of peripheral blood lymphocytes as determined by flow cytometry were minor. In contrast, after immunization a highly significant increase of proliferation in response to stimulation with detoxified toxin A was noted: the stimulation index rose from 1.4 on day 1 to 42.2 on day 74 (restimulation with 0.4 microg/ml; P = 0.003). Immunization led to significant production of interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha by antigen-stimulated lymphocytes. In contrast, no significant induction of interleukin (IL)-4 or IL-10 was observed. In conclusion, immunization of healthy volunteers led to activation of cellular immunity including strong antigen-specific proliferation and cytokine production. In CF patients priming of the cellular immune system towards a Th1-like pattern would be of potential advantage. Therefore, confirmatory analyses in immunized CF patients with and without chronic infection with P. aeruginosa are foreseen.

Cellular immunity in healthy volunteers treated with an octavalent conjugate Pseudomonas aeruginosa vaccine.

Humoral immunity in response to an octavalent O-polysaccharide-toxin A conjugate Pseudomonas aeruginosa vaccine is well studied, and a Phase III clinical study in cystic fibrosis (CF) patients is currently ongoing. In contrast, little is known about cellular immunity induced by this vaccine. Fifteen healthy volunteers were immunized on days 1 and 60. Parameters of cellular immunity were studied before vaccination on day 1, and on day 74. Analyses included flow cytometry of whole blood, and antigen-induced proliferation of and cytokine production by lymphocyte cultures. The effects of immunization on the composition of peripheral blood lymphocytes as determined by flow cytometry were minor. In contrast, after immunization a highly significant increase of proliferation in response to stimulation with detoxified toxin A was noted: the stimulation index rose from 1.4 on day 1 to 42.2 on day 74 (restimulation with 0.4 microg/ml; P = 0.003). Immunization led to significant production of interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha by antigen-stimulated lymphocytes. In contrast, no significant induction of interleukin (IL)-4 or IL-10 was observed. In conclusion, immunization of healthy volunteers led to activation of cellular immunity including strong antigen-specific proliferation and cytokine production. In CF patients priming of the cellular immune system towards a Th1-like pattern would be of potential advantage. Therefore, confirmatory analyses in immunized CF patients with and without chronic infection with P. aeruginosa are foreseen.

Interactions of commensal gut microbes with subsets of B- and T-cells in the murine host.

Although mechanisms operative in the induction and maintenance of specific, adaptive immunity, including 'cognate' B/T interactions, have been extensively studied and defined, we still know little about the mechanisms operative in developing and maintaining B- and T-cell dependent 'natural' immunity. Particularly, we are still rather ignorant concerning gut microbial/gut or systemic APC, T cell and B cell interactions that lead to lymphoid cell mediated 'natural' immunity: specific or broadly reactive, activation via TCR and BCR and/or via other receptors such as the TLR series, and whether T/B interactions are operative at this level? Here we will address: (1) the general role of gut microbes in the development and maintenance of the intestinal, humoral immune system; (2) the general role of gut microbes in the development of B1 cell mediated, 'natural' gut IgA and the dependence of these B1 cells on bystander T cell help; (3) the relative contributions of B1 versus B2 cells to gut 'natural' and specific IgA responses; (4) the role for particular 'normal' gut microbes in the initiation of inflammatory bowel diseases (IBD) in mice with a dysregulated immune system; and (5) the possible roles of gut microbes in facilitating oral tolerance, a mechanism likely operative in forestalling or ameliorating IBD. A central theme of this paper is to attempt to define the specificities of activated, functional CD4+ T cells in the gut for Ags of particular, usually benign gut microbes. We will also consider the still-unresolved issue of whether the contributions of B1-derived IgA in the gut to the 'natural' Ab pool are Ag-selected and driven to proliferation/differentiation or whether the main stimuli are not via BCRs but rather other receptors (TLRs, etc.). The main experimental approach has been to use antigen-free, germ-free, or gnotobiotic (mono- or oligo-associated with precisely known bacterial species) mice.

B2 but not B1 cells can contribute to CD4+ T-cell-mediated clearance of rotavirus in SCID mice.

Studies utilizing various immunodeficient mouse models of rotavirus (RV) infection demonstrated significant roles of RV-specific secretory immunoglobulin A (IgA), CD4+ T cells, and CD8+ T cells in the clearance of RV and protection from secondary infection. Secretion of small but detectable amounts of IgA in RV-infected alphabeta T-cell receptor knockout mice (11) and distinctive anatomical localization and physiology of B1 cells suggested that B1 cells might be capable of producing RV-specific intestinal IgA in a T-cell-independent fashion and, therefore, be responsible for ablation of RV shedding. We investigated the role of B1 cells in the resolution of primary RV infection using a SCID mouse model. We found that the adoptive transfer of unseparated peritoneal exudate cells ablates RV shedding and leads to the production of high levels of RV-specific intestinal IgA. In contrast, purified B1 cells do not ablate RV shedding and do not induce a T-cell-independent or T-cell-dependent, RV-specific IgA response but do secrete large amounts of polyclonal (total) intestinal IgA. Cotransfer of mixtures of purified B1 cells and B1-cell-depleted peritoneal exudate cells differing in IgA allotypic markers also demonstrated that B2 cells (B1-cell-depleted peritoneal exudate cells) and not B1 cells produced RV-specific IgA. To our knowledge, this is the first observation that B1 cells are unable to cooperate with CD4+ T cells and produce virus-specific intestinal IgA antibody. We also observed that transferred CD4+ T cells alone are capable of resolving RV shedding, although no IgA is secreted. These data suggest that RV-specific IgA may not be obligatory for RV clearance but may protect from reinfection and that effector CD4+ T cells alone can mediate the resolution of primary RV infection. Reconstitution of RV-infected SCID mice with B1 cells results in the outgrowth of contaminating, donor CD4+ T cells that are unable to clear RV, possibly because their oligoclonal specificities may be ineffective against RV antigens.

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.

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.

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.

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.