E R de Groot

An IL-13 promoter polymorphism associated with increased risk of allergic asthma

IL-13 plays a crucial role in the development of allergic asthma by several mechanisms, including induction of IgE antibodies, airway eosinophilia and hyper-reactivity. We previously established a deregulated production of IL-13 by T cells from allergic asthma patients. In this report we describe the identification of a novel IL-13 promoter polymorphism (C to T exchange) at position −1055. The IL-13 −1055 TT genotype is associated with allergic asthma (P = 0.002), altered regulation of IL-13 production (P < 0.002), and increased binding of nuclear proteins to this region. we postulate that the presence of this polymorphism predisposes to the development of allergic asthma.

Histamine inhibits the production of interleukin-12 through interaction with H2 receptors.

IL-12 is essential for T helper 1 (Th1) development and inhibits the induction of Th2 responses. Atopic diseases, which are characterized by Th2 responses, are associated with the overproduction of histamine. Here we present evidence that histamine, at physiological concentrations, strongly inhibits human IL-12 p40 and p70 mRNA and protein production by human monocytes. The use of specific histamine receptor antagonists reveals that this inhibition is mediated via the H2 receptor and induction of intracellular cAMP. The inhibition of IL-12 production is independent of IL-10 and IFN-gamma. The observation that histamine strongly reduces the production of the Th1-inducing cytokine IL-12 implies a positive feedback mechanism for the development of Th2 responses in atopic patients.

Imaging and serum analysis of immune complex formation of radiolabelled infliximab and anti-infliximab in responders and non-responders to therapy for rheumatoid arthritis

Background: Many patients with rheumatoid arthritis are currently successfully treated with infliximab (anti-tumour necrosis factor); however, about 30% of the patients do not respond to infliximab. One of the postulated hypotheses of not responding is the fast clearance of infliximab due to the development of infliximab–anti-infliximab complexes. Objective: To investigate the in vivo mechanism of not responding and the role of human anti-chimeric antibodies (HACAs) by using radiolabelled infliximab. Methods: Two responding and two non-responding patients with rheumatoid arthritis, infused with radiolabelled infliximab, were investigated by both imaging and serum analysis. Results: Images showed predominant presence of infliximab in blood up to 24 h, with a trend of faster blood clearance and of higher liver/spleen uptake in a non-responding patient. Clinically inflamed joints showed uptake of the drug. The HACA level in the non-responders was high (1641 and 1008 U/ml), but low or not detectable in responders. Sucrose gradients of serum showed antibody complexes in both non-responders. Various sizes of antibody complexes, including very large ones, were observed in a non-responder who developed a serious infusion reaction. Conclusion: Formation of infliximab–anti-infliximab complexes were found in non-responders due to the presence of large amounts of HACA. This finding, supported by both imaging and serum analysis data, may explain failure of infliximab treatment.

Human intravenous immunoglobulin (IVIG) preparations degranulate human neutrophils in vitro

IVIG preparations have biological effects in vivo that are not fully understood. Possible effects include the property to stimulate Fc receptors on various cell types. To study whether IVIG may interact with neutrophils we developed an in vitro system, in which neutrophils, in whole blood or purified, were incubated with IVIG and assessed for degranulation by measuring the release of elastase and lactoferrin in culture medium. All commercially available IVIG preparations tested induced degranulation of neutrophils when incubated for 2 h at therapeutically relevant concentrations. In studies with blocking antibodies against Fc receptors (FcR), this degranulation was shown to be dependent on FcγRII, whereas FcγRIII had no effect. Experiments with purified neutrophils as well as binding experiments with labelled IVIG preparations indicated that neutrophil degranulation resulted from a direct interaction of IVIG with neutrophils. Using gel filtration fractions, it was found that polymeric and dimeric IgG present in IVIG was mainly responsible for the degranulation. We suggest that degranulation of neutrophils may contribute to the (side)effects of IVIG treatment in vivo.

The antibody response against human and chimeric anti-TNF therapeutic antibodies primarily targets the TNF binding region

Background In a subset of patients, anti tumour necrosis factor (TNF) therapeutic antibodies are immunogenic, resulting in the formation of antidrug antibodies (ADAs). Neutralising ADAs compete with TNF for its binding site and reduces the effective serum concentration, causing clinical non-response. It is however unknown to which extent ADAs are neutralising. Objectives To study which proportion of antibodies to human(ised) anti-TNF (adalimumab, golimumab, certolizumab) as well as chimeric anti-TNF (infliximab) is neutralising. Methods Neutralising capacity of ADAs was assessed using a TNF competition assay in ADA-positive sera of patients treated with adalimumab (n=21), golimumab (n=4), certolizumab (n=9) or infliximab (n=34) sent in to our diagnostic department. Results In 34 sera with ADAs to adalimumab, golimumab or certolizumab, >97% of the antibodies were neutralising. In 34 sera with ADAs to infliximab >90% of the antibodies were neutralising. Further characterisation of the broader antibody response to infliximab revealed that non-neutralising antibodies to infliximab do not target murine domains, but may bind infliximab-unique domains not involved in TNF binding (located outside the paratope). Conclusions Our study shows that ADAs to human(ised) as well as chimeric anti-TNF therapeutic antibodies are largely neutralising. This highly restricted ADA response suggests an immunodominant role for the paratope of anti-TNF therapeutics.

Murine monoclonal isotype switch variants detection with rat monoclonal antibodies in ELISA and isolation by sequential sublining

Isotype switch variants, which arise in monoclonal antibody-producing cell lines, can be detected and selected on the basis of sensitive isotype-specific assays. In this study we used a series of enzyme-linked immunosorbent assays specific for murine IgG1, IgG2b, IgG2a, IgE or IgA, which permitted the detection of low frequency switch variants of hybridoma cell lines, irrespective of the specificity of the secreted antibody. In these assays two rat monoclonal antibodies were combined: one specific for the particular heavy-chain isotype, the other for the light-chain isotype, which was identical in all variants. The value of rat monoclonal antibodies for the detection of isotype switch variants is illustrated by the isolation of a series of variant antibodies specific for the CD3 complex present on human T lymphocytes.

Bioassay for detection of methotrexate in serum

Objective: A bioassay is developed for the measurement of methotrexate (MTX) in serum. Methods: The assay is based on MTX inhibition of the proliferation of hypoxanthine‐guanosine phosphoribosyl transferase (HGPRT) negative mouse B‐cells (B9.H). HGPRT negative cells cannot use the salvage pathway of nucleotide synthesis to overcome inhibition by MTX. Results: When B9.H cells are cultured with serial dilutions of serum, inhibition of proliferation is a measure of the amount of MTX in the serum. Circulating folates do not interfere with the assay. Conclusion: This simple assay can detect low concentrations of MTX in serum: it is therefore useful for following the pharmacodynamics of functional MTX after low‐dose MTX treatment.

Response to: 'The antibody response against human and chimeric anti-TNF therapeutic antibodies primarily targets the TNF binding region’ by Rinaudo-Gaujous et al

Assessment of clinically relevant immunogenicity is a much-debated topic. With respect to the neutralising capacity of antidrug antibodies (ADAs) to tumour necrosis factor (TNF) blockers, we recently demonstrated that the vast majority of antibodies in fact compete with TNF binding.1 Therefore, we are pleased to find this notion further supported by the letter of Rinaudo-Gaujous et al .2 The aim of our study was to quantify to which extent ADAs were neutralising. Using the TNF competition assay, we concluded that …

The effect of methotrexate and mycophenolic acid on monokine production in vitro

Methotrexate (MTX) and mycophenolic acid (MPA) are used clinically for their immunosuppressive properties. MTX is widely used for the treatment of RA. MPA is used to prevent graft rejection and is now experimentally used in SLE and RA. The precise mechanism of action is still debated. Both drugs, though in different ways, inhibit the de novo synthesis of DNA and RNA. We have analysed cytokine production in short cell cultures in whole blood and isolated cells by ELISA. We have shown before that both drugs inhibit the production of several cytokines after T-cell stimulation, and we concluded that MTX leads to irreversible elimination of activated T cells by apoptosis, whereas MPA reversibly prevents activation of resting T cells. We now show that when monocytes are stimulated by SAC or lipopolysaccharide, both MTX and MPA decrease TNF-α, IL-6 and IL-8 production. However the inhibition is not as profound as after T-cell stimulation. An exception is the effect on the production of the proinflammatory cytokine IL-1β. The production of IL-1β is not influenced by MTX after SAC or LPS stimulation, whereas the production is increased by MPA when cells are stimulated with LPS, but not with SAC. We have investigated the cause for this increase. The expression of IL-1β mRNA is not influenced by MPA. Immunoprecipitation and ELISA show that MPA leads to a decrease in the intracellular proform of IL-1β. We conclude that MPA leads to enhanced cleavage of pro-IL-1β.