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Background: Human intravenous immunoglobulin (IVIg) preparations are used for the treatment of autoimmune and allergic diseases. Natural autoantibodies are believed to contribute to IVIg-mediated anti-inflammatory effects. Objective: To address the question of whether IVIg preparations contain anti–sialic acid-binding Ig-like lectin-8 (anti–Siglec-8) autoantibodies. Methods: The presence of possible anti–Siglec-8 autoantibodies in IVIg preparations was first examined by functional eosinophil death and apoptosis assays. Specificity of IVIg effects was shown by depleting anti–Siglec-8 autoantibodies from IVIg. Binding of purified anti–Siglec-8 autoantibodies to recombinant Siglec-8 was demonstrated by an immunodot assay. Results: IVIg exerts cytotoxic effects on purified human blood eosinophils. Both potency and efficacy of the IVIg-mediated eosinophil killing effect was enhanced by IL-5, granulocyte/macrophage colony-stimulating factor, IFN-γ, TNF-α, and leptin. Similarly, inflammatory eosinophils obtained from patients suffering from the hypereosinophilic syndrome (HES) demonstrated increased Siglec-8 cytotoxic responses when compared with normal blood eosinophils. Pharmacologic blocking experiments indicated that the IVIg-mediated additional eosinophil death in the presence of cytokines is largely caspase-independent, but it depends on reactive oxygen species. Anti–Siglec-8 autoantibody-depleted IVIg failed to induce caspase-independent eosinophil death. Conclusion: IVIg preparations contain natural anti–Siglec-8 autoantibodies. Clinical implications: Anti–Siglec-8 autoantibodies present in IVIg preparations may have therapeutic relevance in autoimmune and allergic diseases, respectively, such as Churg-Strauss syndrome. [Copyright &y& Elsevier]

Atopic diseases such as asthma, rhinitis, eczema and food allergies have increased in most industrialised countries of the world during the last 20 years. The reasons for this increase are not known and different hypotheses have been assessed including increased exposure to sensitising allergens or decreased stimulation of the immune system during critical periods of development.In allergic diseases there is a polarisation of the Th2 response and an increase in the production of type 2 cytokines which are involved in the production of immunoglobulin E and the development of mast cells, basophils and eosinophils leading to inflammation and disease. The effector phase of atopy is initiated by interaction with FcϵRI expressed on effector cells such as mast cells and basophils but also found on an ever increasing list of cells. Binding of a polyvalent allergen to the variable part of IgE leads to a cross-link of the receptor that triggers the cell to release histamine and pharmacological mediators of the symptomatic allergic response. Cross-linking of FcϵRI by autoantibodies against the α-chain of the FcϵRI, causing subsequent histamine release is thought to be involved in the pathogenesis of other diseases such as chronic idiopathic urticaria (CIU).To date, most therapeutic strategies are aimed at inhibiting and controlling components of the inflammatory response. Recently, new treatment strategies have emerged that focus on the development of preventive and even curative treatments. The most promising therapeutic approaches are aimed at inhibiting the IgE–FcϵRI interaction with the use of non-anaphylactogenic anti-IgE or anti-FcϵRIα autoantibodies. Clinical trials in humans using an humanised anti-IgE antibody showed that this antibody was well tolerated and reduced both symptoms and use of medication in asthma and allergic rhinitis. Thus interruption of the atopic cascade at the level of the IgE–FcϵRI interaction with the use of non-anaphylactogenic antibodies is effective and represents an attractive therapy for the treatment of atopic disease. [Copyright &y& Elsevier]

Background: Specificities for carbohydrate IgG antibodies, thought to be predominantly of the IgG2 subclass, have never been broadly examined in healthy human subjects. Objective: To examine commercial intravenous immunoglobulin (IVIG) preparations for their ability to recognize a wide range of glycans and to determine the contribution of IgG2 to the binding pattern observed. Methods: We used a glycan microarray to evaluate IVIG preparations and a control mix of similar proportions of human myeloma IgG1 and IgG2 for binding to 377 glycans, courtesy of the Consortium for Functional Glycomics Core H. Glycans recognized were categorized using public databases for their likely cellular sources. IgG2 was depleted from IVIG by using immunoaffinity chromatography, and depletion was confirmed by using nephelometry and surface plasmon resonance. Results: Nearly half of the glycans bound IgG. Some of the glycans with the greatest antibody binding can be found in structures of human pathogenic bacteria (eg, Streptococcus pneumoniae, Mycobacterium tuberculosis, Vibrio cholera) and nonpathogenic bacteria, including LPS and lipoteichoic acid, capsular polysaccharides, and exopolysaccharides. Surprisingly, depletion of IgG2 had only a modest effect on anticarbohydrate recognition patterns compared with the starting IVIG preparation. Little to no binding activity was detected to human endogenous glycans, including tumor-associated antigens. Conclusions: This novel, comprehensive analysis provides evidence that IVIG contains a much wider range than previously appreciated of anticarbohydrate IgG antibodies, including those recognizing both pathogenic and non–pathogen-associated prokaryotic glycans. [Copyright &y& Elsevier]

Intravenous administration of polyclonal and monoclonal antibodies has proven to be a clinically valid approach in the treatment, or at least relief, of many acute and chronic pathologies, such as infection, immunodeficiency, and a broad range of autoimmune conditions. Plasma-derived IgG or recombinant IgG are most frequently used for intravenous or subcutaneous administration, whereas a few IgM-based products are available as well.Wehave established recently that secretory-like IgA and IgM can be produced upon association of plasma-derived polymeric IgA and IgM with a recombinant secretory component. As a next step toward potential future mucosal administration, we sought to unravel the mechanisms by which these secretory Igs protect epithelial cells located at the interface between the environment and the inside of the body. By using polarized epithelial Caco-2 cell monolayers and Shigella flexneri as a model enteropathogen, we found that polyspecific plasma-derived SIgA and SIgM fulfill many protective functions, including dose-dependent recognition of the antigen via formation of aggregated immune complexes, reduction of bacterial infectivity, maintenance of epithelial cell integrity, and inhibition of proinflammatory cytokine/chemokine production by epithelial cells. In this in vitro model devoid of other cellular or molecular interfering partners, IgM and secretory IgM showed stronger bacterial neutralization than secretory IgA. Together, these data suggest that mucosally delivered antibody preparations may be most effective when combining both secretory-like IgA and IgM, which, together, play a crucial role in preserving several levels of epithelial cell integrity. [ABSTRACT FROM AUTHOR]

Immunotherapy with monoclonal and polyclonal immunoglobulin is successfully applied to improve many clinical conditions, including infection, autoimmune diseases, or immunodeficiency. Most immunoglobulin products, recombinant or plasma-derived, are based on IgG antibodies, whereas to date, the use of IgA for therapeutic application has remained anecdotal. In particular, purification or production of large quantities of secretory IgA (SIgA) for potential mucosal application has not been achieved. In this work, we sought to investigate whether polymeric IgA (pIgA) recovered from human plasma is able to associate with secretory component (SC) to generate SIgA-like molecules. We found that ~15% of plasma pIgA carried J chain and displayed selective SC binding capacity either in a mixture with monomeric IgA (mIgA) or after purification. The recombinant SC associated covalently in a 1:1 stoichiometry with pIgA and with similar efficacy as colostrum-derived SC. In comparison with pIgA, the association with SC delayed degradation of SIgA by intestinal proteases. Similar results were obtained with plasma-derived IgM. In vitro, plasma-derived IgA and SIgA neutralized Shigella flexneri used as a model pathogen, resulting in a delay of bacteria-induced damage targeted to polarized Caco-2 cell monolayers. The sum of these novel data demonstrates that association of plasma-derived IgA or IgM with recombinant/colostrum-derived SC is feasible and yields SIgA- and SIgM-like molecules with similar biochemical and functional characteristics as mucosa-derived immunoglobulins. [ABSTRACT FROM AUTHOR]

Abstract: Intravenous immunoglobulin (IVIg) preparations are derived from pooled plasma from up to 60,000 healthy human donors and reflect the immunologic experience of the donor population. IVIg contains monomeric and dimeric IgG populations which are in a dynamic equilibrium depending on concentration, pH, temperature, donor pool size, time and stabilizers added in order to keep the portion of dimeric IgG below a certain level. In the present study, monomeric and dimeric fractions were isolated by size exclusion chromatography. The dimeric fractions, however, showed a dynamic instability and tended to dissociate. Both dimeric and monomeric IgG fractions were acid treated (pH 4) in order to dissociate the dimeric IgG. Western-blot analysis identified a sub-population of SDS resistant IgG dimers. Furthermore, the reactivities of the fractions were tested against a panel of self- and exo-antigens. There was a marked increase in activity of the dimeric compared to the monomeric IgG fraction against various intracellular self-antigens. Our data indicates that the increased reactivities of pH 4-treated fractions can mainly be attributed to dimer dissociation, as pH 4-treated monomers do not show significantly increased activities against a range of antigens. [Copyright &y& Elsevier]

Abstract: Intravenous immunoglobulin (IVIG) is involved in many complex mechanisms that act in synergy including expression and function of Fc receptors, complement activation, the cytokine network, interaction with the anti-idiotypic network and modulation of B and T cell activation. To gain insight into the early effects of IVIG on this broad range of activities at the gene level we performed DNA microarray analysis. Human whole blood was incubated in vitro for 4h followed by extraction of RNA which was hybridized to a chip containing 8793 genes. About 75 upregulated genes and 21 downregulated genes were identified using a cut off for the false discovery rate of 5%. These genes are associated with a wide range of cellular immune functions in line with the broad mechanism of action of IVIG. A striking upregulation of a series of genes coding for chemokines was measured. This finding was confirmed at the protein level as pharmacologically relevant concentrations of CXCL9 and CXCL10 were measured in serum. Interestingly, IVIG shows a partial overlap of its gene expression program with lipopolysaccharide. Our data suggests multiple hypotheses regarding the pharmacology of IVIG that must be validated by complementary studies. [Copyright &y& Elsevier]

Abstract: We have previously isolated anti-FcεRIα autoantibodies from phage libraries of healthy donors and urticaria patients. Strikingly, the same antibody, LTMα15, was isolated from both libraries. Sequence analysis revealed a germline configuration of the LTMα15 variable heavy (VH) chain with a slightly mutated variable light (VL) chain supporting its classification as a natural autoantibody. Distribution analysis of anti-FcεRIα autoantibodies by functional or serological tests delivered conflicting data. For this reason we have developed a new real-time PCR to analyse the distribution of LTMα15VH in healthy donors and urticaria patients. Our new bioinformatic program permitted the design of a minor groove binder (MGB) TaqMan probe that specifically detected the LTMα15VH. We were able to demonstrate a broad range of rearranged VH gene copy number without any correlation to the state of health. Monitoring LTMα15VH gene copy number in a single donor over a period of 70 days revealed a time-related fluctuation of circulating B cells carrying LTMα15VH. We propose that our real-time PCR may serve as a model for the quantification of natural antibody sequences at a monoclonal level. [Copyright &y& Elsevier]

Abstract: We have previously demonstrated the induction of a specific anti-IgE response in vivo by parenteral immunisation of rhesus monkeys using short IgE mimotopes or an anti-idiotypic antibody mimicking an IgE epitope. Such specific anti-IgE responses may be of clinical benefit for atopic patients. In this study, we examined the potential for a more convenient therapy via mucosal immunisation using live recombinant Lactobacillus johnsonii (Lj) as a vaccine delivery vehicle. Either an anti-idiotypic scFv or an IgE mimotope were expressed on the surface of Lj as fusion proteins using the cell wall anchored proteinase PrtB from Lactobacillus delbrueckii subsp. bulgaricus. The recombinant Lj were shown to express the heterologous fusion proteins and were specifically recognised by the corresponding anti-human IgE monoclonal antibody. Subcutaneous and intranasal immunisation of mice with recombinant Lj, expressing these fusion proteins induced a systemic IgG response against human IgE. Our data suggest that recombinant Lactobacilli expressing IgE epitopes may represent a novel means of vaccination to induce a beneficial anti-IgE response. [Copyright &y& Elsevier]

Lactobacilli are considered to be safe organisms making them attractive as vehicles for oral vaccination. We report that Lactobacillus johnsonii (Lj) partially survived simulated gastric conditions in vitro, suggesting that it could be used as an oral vaccine delivery vehicle. In order to test this approach, we used the cell wall anchored proteinase PrtB, isolated from Lactobacillus delbrueckii subsp. bulgaricus as a model antigen. Using a new vector system, we demonstrated expression of both proteinase PrtB alone and a mimotope peptide derived from tetanus toxin integrated in the sequence of proteinase PrtB (TTmim–PrtB fusion protein) on the surface of Lj. Oral immunisation of mice with recombinant Lj, expressing the TTmim–PrtB fusion protein induced a systemic IgG response against Lj and recombinantly expressed proteinase PrtB but no antibody response against the tetanus toxin mimotope suggesting that the mimotope was not sufficiently immunogenic to induce an immune response. Interestingly, a proteinase PrtB specific fecal IgA response was also induced, indicating that the proteinase PrtB fusion protein expressed as a cell surface protein on Lj can induce both systemic and local mucosal immune responses. [Copyright &y& Elsevier]