Els R. de Groot

IMMUNOLOGY OF DNA. III. CRITHIDIA LUCILIAE, A SIMPLE SUBSTRATE FOR THE DETERMINATION OF ANTI-dsDNA WITH THE IMMUNOFLUORESCENCE TECHNIQUE*

C. luciliae are hemoflagellates nonpathogenic for man and easy to culture. They have a giant mitochondrion, in which the mitochondrial DNA is concentrated in a single large network, the kinetoplast. When used as a substrate for the indirect immunofluorescence technique, studying sera from patients with SLE, we could demonstrate a very good correlation between this test and the Farr assay for the demonstration of antibodies to double-stranded DNA. Although the sensitivity of both techniques is on the same order of magnitude, the IF technique has the following advantages over the Farr assay. It is easy to perform in laboratories equipped for autoimmune serology. It possesses an intrinsic check on the immunoglobulin character of the DNA-binding activity. It allows one to determine the Ig classes and subclasses of antibodies to DNA. It permits study of complement fixation to antibodies without interference of Clq fixation to DNA or anticomplementarity of the serum. There is an absence of interference with antibodies to single-stranded DNA.

Sensitive ELISA for interleukin-6. Detection of IL-6 in biological fluids: synovial fluids and sera.

A monoclonal antibody and an affinity purified polyclonal antibody, both raised against recombinant human IL-6, have been employed in an ELISA procedure to quantitate human IL-6. Both antibodies were very potent in neutralizing the biological activity of recombinant as well as natural human IL-6. The monoclonal antibody was used as the capture antibody whilst the polyclonal antibody, in biotinylated form, was used as the detecting antibody in combination with a streptavidin horseradish peroxidase conjugate and a signal amplification system. The detection limit for natural as well as recombinant IL-6 was 1 pg/ml. A good correlation was found between the ELISA and the B9 biological assay when IL-6 was measured in crude culture supernatants, in synovial fluids of rheumatoid arthritis patients and in the sera of patients with diverse diseases. Immunoprecipitation of IL-6, produced by different cell types, such as monocytes, endothelial cells and smooth muscle cells or derived from biological fluids, such as the serum of a patient with septic shock or the synovial fluid of a rheumatoid arthritis patient, revealed in every case only molecules in the molecular weight range of 21,000-26,000.

Adalimumab elicits a restricted anti-idiotypic antibody response in autoimmune patients resulting in functional neutralisation

Objectives Millions of patients worldwide are treated with therapeutic monoclonal antibodies. These biological therapeutics can be immunogenic, resulting in anti-drug antibody formation which leads to loss of response. Fully human biological agents, such as the anti-tumour necrosis factor α (anti-TNFα) antibody adalimumab, are considered to be weakly immunogenic, but anti-adalimumab antibodies (AAA) were recently detected in more than half of treated patients with rheumatoid arthritis (RA) within 28 weeks of treatment. A study was undertaken to determine the mechanism by which AAA lead to loss of response. Methods The specificity of the repertoire of AAA was investigated in a cohort of 50 AAA-positive RA patients. Inhibition experiments using TNFα and patient-derived anti-adalimumab monoclonal antibodies were performed. Results The antibody response against adalimumab is highly restricted: Fab fragments of a single monoclonal antibody specific for the idiotype of adalimumab inhibited 98.65% (25th–75th percentiles: 98.25–99.90) of the total anti-adalimumab reactivity in serum from 50 AAA-positive patients. The anti-adalimumab response was confined to the TNFα binding region of adalimumab, thereby neutralising its therapeutic efficacy. In line with this restricted specificity, small immune complexes were found in the circulation of AAA-forming patients. Conclusions The humoral immune response against adalimumab is highly restricted and limited to the idiotype of the therapeutic antibody. All antibodies result in functional neutralisation of the drug, thereby providing a mechanism by which AAA formation leads to clinical non-response.

Differential effect of drug interference in immunogenicity assays

The presence of anti-drug antibodies (ADA) in adalimumab-treated patients is associated with reduced serum adalimumab levels and a lower clinical response. Currently, there is no standard for measurement of anti-drug antibodies and many factors influence the results. Consequently, the incidence of ADA as reported in different studies varies considerably. Here we investigated the differential effect of drug interference in two common types of assays used to measure anti-adalimumab: an antigen binding test (ABT) and a more often-used bridging elisa. We measured ADA to adalimumab in a cohort of 216 rheumatoid arthritis patients treated with adalimumab for 28 weeks. Only 15 samples (7%) were positive in the bridging elisa, compared to 29 (13%) in the ABT, despite the fact that the bridging elisa was the most sensitive assay. Furthermore, in an ABT specific for IgG4, 48 samples (22%) were found positive. The bridging elisa was found to detect only the bivalent form of (drug-specific) IgG4, resulting in an underestimation of ADA levels. However, the predominant reason for the different outcomes of these assays was a differential susceptibility to drug interference. In particular, the bridging elisa only detected ADA in the absence of detectable amounts of circulating adalimumab and is therefore not suited for measurement of ADA in complex with the drug. In summary, we showed that a bridging elisa is susceptible to drug interference and typically measures ADA only in absence of detectable drug levels.

Immunology of DNA. IV. Quantitative aspects of the Farr assay

We have investigated whether the Farr assay measures the primary interaction between DNA and anti-DNA. By the use of zonal centrifugation to analyse DNA/anti-DNA complexes and by a detailed study of the quantitative aspects of the Farr assay we demonstrate that: (a) binding of a single IgG molecule to a PM2 DNA molecule (molecular weight 5.9 X 10(6)) renders the latter precipitable in half-saturated ammonium sulphate; (b) two antibody molecules are required for precipitation of T7 DNA (molecular weight 25 X 10(6)); (c) millipore filtration detects binding of a single antibody molecule per molecule of PM2 DNA; (d) theoretically and experimentally the relation between free DNA (F) and the amount of serum (S) can be described by IfF =-cS where c is a constant; (e) it is feasable to quantitate serum anti-DNA activity in terms of units per ml.

Potentiation of Toll-like receptor-induced cytokine production by (1→3)-β-D-glucans: implications for the monocyte activation test

The monocyte activation test (MAT) has been introduced as an alternative for the detection of pyrogens in pharmaceuticals with the rabbit pyrogen test or the Limulus amebocyte lysate (LAL) test. The basis of the MAT is that pyrogens, via Toll-like receptors (TLRs) expressed on monocytes, stimulate cytokine production. Here, we report that, at concentrations that did not induce whole blood cytokine production when tested separately, (1→3)-β-D-glucans powerfully co-stimulated cytokine production (IL-6/IL-8) induced by ligands for TLR1/2, TLR2/6, TLR4, and TLR5. Experiments were performed to investigate the involvement of particular (1→3)-β-D-glucan receptors such as dectin-1. Spleen tyrosine kinase (Syk) inhibition attenuated the potentiating effects of (1→3)-β-D-glucans on TLR-induced cytokine production, suggesting that dectin-1 was involved. However, experiments with low molecular (1→3)-β-D-glucans such as laminarin argued against the involvement of dectin-1 in the co-stimulatory effects of (1→3)-β-D-glucans. Thus, although the receptors involved in the co-stimulatory actions of (1→3)-β-D-glucans on TLR-induced cytokine production are yet to be elucidated, it is clear that (1→3)-β-D-glucans may greatly affect MAT results and, when undetected in pharmaceuticals, may give rise to serious side-effects in patients co-exposed to other elicitors of innate immunity, such as during infections.

Cytokine induction by pyrogens: comparison of whole blood, mononuclear cells, and TLR-transfectants

Given the shortcomings in the measurement of pyrogenic contamination of pharmaceuticals and/or test substances by means of the rabbit pyrogen test and the Limulus amoebocyte lysate (LAL) test, several in vitro pyrogen tests have been developed based on the measurement of cytokine production by monocytes. In this study we measured cytokine production (IL-6, IL-8, IL-1beta, and TNF) in diluted whole blood (WB), mononuclear cells (MNC), and HEK cells stably transfected with CD14 and Toll-like Receptor-2 (TLR2) or TLR4, after stimulation with both standard pyrogens and contaminated substances. Our study demonstrated that in MNC, IL-6 production was more sensitive to pyrogen stimulation than IL-1beta and TNF production. The sensitivity of WB IL-8 production for pyrogens was comparable with that of MNC IL-6 production, but higher than WB IL-6 production. MNC IL-8 production as readout for pyrogenic stimulation was not useful due to high background IL-8 production. Surprisingly, contaminated culture media potently stimulated WB IL-8 production, but not MNC IL-6 production. Finally, the value of TLR-transfected HEK cells in the detection of pyrogenic contamination as well as the role of IL-10 in interindividual differences in cytokine production, is discussed. To summarize, the results presented herein together with literature data indicate that the measurement of WB IL-8 production may represent an advantageous alternative to the measurement of MNC IL-6 production, for the detection of pyrogenic contamination of pharmaceuticals.

Immunology of DNA. V. Analysis of DNA/anti-DNA complexes

DNA/anti-DNA complexes were studied by equilibrium density centrifugation in CsC1 and by zonal centrifugation in sucrose gradients. Complexes prepared of circular DNA preparations PM2 DNA or SV40 DNA with anti-DNA obtained from patients with systemic lupus erythematosus were found to be stable under the conditions of CsC1 equilibrium centrifugation. This enabled homogeneous complexes of DNA and anti-DNA of composition AgAb1, AgAb2, AgAb3 etc to be isolated. The stability of these complexes, their banding pattern in CsC1 gradients and their sedimentation behaviour suggest that anti-DNA binds to DNA in a monogamous bivalent way.

Immunology of DNA. VI. The effect of mercaptans on IgG and IgM anti-dsDNA

Reduction of IgM antibodies to DNA with mercaptans such as dithioerythrol or 2-mercaptoethanol completely destroys DNA binding in the Farr assay and in the immunofluorescence technique by Crithidia luciliae. In contrast reduction of IgG antibodies to DNA results in a six-fold increase of DNA binding in the Farr assay while no effect on titres in the immunofluorescence technique can be observed. Our results lead to the following conclusions: 1) The Farr assay is selective for high avidity interactions; only a minor part of IgG antibodies to DNA is measured; 2) 7S IgM antibodies to DNA cannot be demonstrated in the Farr assay or the immunofluorescence technique; obviously only multivalent interactions, as obtained with the intact 19S IgM molecule are stable in these assays; 3) reduction of IgG leads to a greater flexibility of this molecule; this facilitates monogamous bivalent binding to DNA; 4) THE PRESENTATION OF DNA in the kinetoplast of Crithidia luciliae favours a monogamous bivalent binding of antibodies to DNA with high avidity; this accounts for occasionally observed discrepancies between anti-DNA activity in the Farr assay and in the immunofluorescence technique.

Changes of ferritin and CRP levels in melanoma patients treated with adjuvant interferon-α (EORTC 18 952) and prognostic value on treatment outcome

Adjuvant therapy with interferon-&agr; (IFN) only benefits a small subgroup of melanoma patients and a predictive marker selecting responders does not exist. IFN induces increased ferritin and decreased C-reactive protein (CRP) levels; however, an association with treatment effect was not studied. Serum was collected from patients participating in the European Organization for Research and Treatment of Cancer 18 952 trial comparing adjuvant treatment with IFN to observation. Serial ferritin and CRP levels were determined using enzyme-linked immusorbent assays, before treatment and up to 24 months. Ferritin levels are influenced by sex and age; therefore ratios of serial ferritin and CRP values with corresponding pretreatment values were calculated. Cox regression model and landmark method at end of induction and 6 months were used to evaluate the association between ferritin, CRP and distant metastasis-free survival (DMFS). Baseline ferritin levels were comparable in the two treatment groups (P=0.92). However, ferritin ratios were significantly higher in IFN-treated patients (N=96) compared with untreated patients (N=21) at end of induction (mean: 2.88 vs. 0.75; P=0.0003) and at 6 months (mean: 3.18 vs. 1.02; P=0.009). In the IFN arm, higher ferritin ratios at end of induction and at 6 months were not associated with improved outcome (respectively, P=0.66 and 0.86). Concerning CRP ratios, no differences between the treatment groups, neither an association with DMFS, were observed. Administration of IFN in melanoma patients induced increase in ferritin levels but not in CRP levels. Ferritin and CRP ratios have no prognostic value regarding DMFS.