Induction and measurement of IgE : a study in mice, with emphasis on the regulatory role of lymphokines

A better understanding of the regulatory mechanisms and cellular interactions of the IgE antibody response is of fundamental interest to allergologists and clinical immunologists because of the role of IgE in the pathogenesis of the immediate type allergic disease. In addition, basic knowledge of the regulation of IgE antibody formation may lead to new forms of treatment including suppression of excessively formed IgE antibody. Beyond the clinical significance, the IgE antibody response provides an excellent model to establish the interdependencies and regulatory events governing the expression of different isotypes at different levels. The formation of IgE antibodies is regulated by T cells and controlled by antigen- and/or isotype-specific interactions. Thus, external modulation of IgE production can be achieved by antigen, idiotype and anti-idiotype, being specific recognition elements in the establishment and control of an immune response. Also IgE specific regulatory factors and receptors on different types of cells exert a regulatory influence. In the case of isotype-specific regulation, total IgE antibody may be affected irrespective of its specificity. This is of relevance for eventual treatment of generalized IgE mediated allergic diseases. On the other hand, an antigen-specific modulation of an immune response will also affect the expression of other isotypes, even in a secondary response. From the above it is clear that much of the knowledge on the induction of the allergic inflammation gained from animal studies is clinically relevant. Moreover, the basic mechanisms of IgE regulation seem to be similar in man and in mice and rats. This is the underlying premise for these studies. The purpose of the investigations presented in this thesis was to get more insight into the mechanisms underlying the induction of IgE antibody formation in the mouse and the regulation of this IgE synthesis. For this study the development of suitable reagents and appropriate assay systems was indicated. Only since the availability of antigen-specific mouse IgE-secreting hybridomas, it became feasible to isolate enough IgE for the induction of heterologous anti-IgE antisera. This purified IgE can also be used as a reference standard in the quantitative determination of IgE. Furthermore, the hybridoma cells can be employed for the standardization of techniques that allow the determination of IgE-secreting cells. It is therefore that in Chapter 4 we focuss on the isolation and purification of monoclonal murine IgE, the generation of heterologous IgE-specific antisera and the development of a Terasaki-ELISA system for the quantitative determination of secreted IgE. Employing one of these an

Semi-preparative purification and validation of monoclonal antibodies for immunotherapy in mice

A number of rat hybridomas were adapted to grow in RPMI containing either 5% IgG-depleted FCS of 1% serum-free Nutridoma. Alternatively, protein-free Ultradoma PF was used. Growth in these media allowed purification procedures to be used that are based on tangential ultrafiltration in combination with affinity chromatography on gels linked to protein G or anti-rat L chain coupled antibodies. The isolated antibody preparations were found to be pure and to consist of monomeric intact IgG. The yield and recovery of mAb using this procedure were found to be consistently high. These antibody preparations were analyzed for endotoxin contamination. Whereas during isolation endotoxin contamination increased, the endotoxin content per mg purified protein did not. Affinity chromatography on Detoxi-gel resulted in the efficient removal of this contamination and using this protocol the antibody preparations obtained were found to be of sufficient purity, activity and low endotoxin content to permit their in vivo use in animal models of immunotherapy

Modulation of systemic cytokine levels by implantation of alginate encapsulated cells

The availability of cell lines that are transfected with IL-4, IL-5 and IFN-γ cytokine genes permits the prolonged in vivo delivery of functional cytokines in relatively large doses for the modulation of specific immune responses. Oft

Involvement of T cells in enhanced resistance to Klebsiella pneumoniae septicemia in mice treated with liposome-encapsulated muramyl tripeptide phosphatidylethanolamine or gamma interferon

We have previously shown that prophylactic administration of the liposome-encapsulated immunomodulating agents muramyl tripeptide phosphatidylethanolamine (MTPPE) and gamma interferon (IFN-gamma) results in strongly increased survival of mice from a normally lethal septicemia with Klebsiella pneumoniae. It was anticipated that the treatment acts on macrophages and nonspecifically augments host resistance to various infections. In the present study, we provide evidence for a key role for T cells in host defense potentiation by the liposomal immunomodulators toward K. pneumoniae septicemia. It is shown that both CD4 and CD8 cells are important in immunomodulation, most likely due to production of IFN-gamma. Depletion of circulating IFN-gamma resulted in strong reduction of the antimicrobial host defense activation. Administration of interleukin-10 resulted in decreased antimicrobial host defense activation by liposomal immunomodulators. Moreover, administration of liposomal immunomodulators was shown to induce predominantly T-helper type 1 (Th1) cell populations in the spleen. These findings indicate that immunomodulation with liposomal MTPPE and IFN-gamma favors Th1 and NK cell activation

Fc receptor binding of anti-CD3 monoclonal antibodies is not essential for immunosuppression, but triggers cytokine-related side effects

A major drawback to the use of OKT3, a mouse anti-CD3 monoclonal antibody (mAb), as an immunosuppressive agent is the associated cytokine release syndrome. We used a mouse model to elucidate the properties of anti-CD3 mAb responsible for these cytokine-related side effects. We have previously demonstrated that the hamster anti-CD3 mAb 145-2C11 induced strong cytokine release and morbidity in vivo, whereas two rat anti-CD3 mAb 17A2 and KT3 did not. In the current study, we show that the mitogenic capacity of soluble anti-CD3 mAb in vitro correlates with their induction of side effects in vivo. Mitogenesis in vitro and tumor necrosis factor-α (TNF-α) release in vivo induced by anti-CD3 mAb could be inhibited by the anti-FcγR mAb 2.4G2, indicating that FcγR binding of anti-CD3 mAb is responsible for their mitogenic properties and for their induction of side effects. Importantly, the two non-mitogenic rat anti-CD3 mAb were equally capable of suppressing skin allograft rejection as the mitogenic hamster anti-CD3 mAb, suggesting FcγR binding of anti-CD3 mAb is not essential for their immunosuppressive properties. This suggestion is reinforced by our demonstration that administration of 2.4G2 in vivo did not interfere with immunosuppression of skin allograft rejection by 145-2C11. These findings suggest that clinical use of non-mitogenic anti-CD3 mAb will result in effective immunosuppression without cytokine-related side effects

Binding of CML-Modified as Well as Heat-Glycated beta-lactoglobulin to Receptors for AGEs Is Determined by Charge and Hydrophobicity

Intake of dietary advanced glycation end products (AGEs) is associated with inflammation-related health problems. Nε-carboxymethyl lysine (CML) is one of the best characterised AGEs in processed food. AGEs have been described as ligands for receptors present on antigen presenting cells. However, changes in protein secondary and tertiary structure also induce binding to AGE receptors. We aimed to discriminate the role of different protein modifications in binding to AGE receptors. Therefore, β-lactoglobulin was chemically modified with glyoxylic acid to produce CML and compared to β-lactoglobulin glycated with lactose. Secondary structure was monitored with circular dichroism, while hydrophobicity and formation of β-sheet structures was measured with ANS-assay and ThT-assay, respectively. Aggregation was monitored using native-PAGE. Binding to sRAGE, CD36, and galectin-3 was measured using inhibition ELISA. Even though no changes in secondary structure were observed in all tested samples, binding to AGE receptors increased with CML concentration of CML-modified β-lactoglobulin. The negative charge of CML was a crucial determinant for the binding of protein bound CML, while binding of glycated BLG was determined by increasing hydrophobicity. This shows that sRAGE, galectin-3, and CD36 bind to protein bound CML and points out the role of negatively charged AGEs in binding to AGE receptors

Multicentre double-blind placebo-controlled food challenge study in children sensitised to cashew nut

Background: Few studies with a limited number of patients have provided indications that cashew-allergic patients may experience severe allergic reactions to minimal amounts of cashew nut. The objectives of this multicentre study were to assess the clinical relevance of cashew nut sensitisation, to study the clinical reaction patterns in double-blind placebo-controlled food challenge tests and to establish the amount of cashew nuts that can elicit an allergic reaction. Methods and Findings: A total of 179 children were included (median age 9.0 years; range 2-17 years) with cashew nut sensitisation an

IgE cross-reactivity measurement of cashew nut, hazelnut and peanut using a novel IMMULITE inhibition method

Tree nut-allergic individuals are often sensitised towards multiple nuts and seeds. The underlying cause behind a multi-sensitisation for cashew nut, hazelnut, peanut and birch pollen is not always clear. We investigated whether immunoglobulin E antibody (IgE) cross-reactivity between cashew nut, hazelnut and peanut proteins exists in children who are multi-allergic to these foods using a novel IMMULITE®-based inhibition methodology, and investigated which allergens might be responsible. In addition, we explored if an allergy to birch pollen might play a role in this co-sensitisation for cashew nut, hazelnut and peanut. Serum of five children with a confirmed cashew nut allergy and suffering from allergic symptoms after eating peanut and hazelnut were subjected to

IgE Cross-Reactivity of Cashew Nut Allergens

Background: Allergic sensitisation towards cashew nut often happens without a clear history of eating cashew nut. IgE cross-reactivity between cashew and pistachio nut is well described; however, the ability of cashew nut-specific IgE to cross-react to common tree nut species and other Anacardiaceae, like mango, pink peppercorn, or sumac is largely unknown. Objectives: Cashew nut allergic individuals may cross-react to foods that are phylogenetically related to cashew. We aimed to determine IgE cross-sensitisation and cross-reactivity profiles in cashew nut-sensitised subjects, towards botanically related proteins of other Anacardiaceae family members and related tree nut species. Method: Sera from children with a suspected cashew nut allergy (n = 56) were assessed for IgE sensitisation to common tree nuts, mango, pink peppercorn, and sumac using dot blo