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.
To study which proportion of antibodies to human(ised) anti-TNF (adalimumab, golimumab, certolizumab) as well as chimeric anti-TNF (infliximab) is neutralising.
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.
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).
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.
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.
Oncogene-induced cellular senescence (OIS) is emerging as a potent cancer-protective response to oncogenic events, serving to eliminate early neoplastic cells from the proliferative pool. Using ...combined genetic and bioinformatic analysis, we find that OIS is linked specifically to the activation of an inflammatory transcriptome. Induced genes included the pleiotropic cytokine interleukin-6 (IL-6), which upon secretion by senescent cells acted mitogenically in a paracrine fashion. Unexpectedly, IL-6 was also required for the execution of OIS, but in a cell-autonomous mode. Its depletion caused the inflammatory network to collapse and abolished senescence entry and maintenance. Furthermore, we demonstrate that the transcription factor C/EBPβ cooperates with IL-6 to amplify the activation of the inflammatory network, including IL-8. In human colon adenomas, IL-8 specifically colocalized with arrested, p16
INK4A-positive epithelium. We propose a model in which the context-dependent cytostatic and promitogenic functions of specific interleukins contribute to connect senescence with an inflammatory phenotype and cancer.
Immunogenicity, specifically the onset of antibodies against tumour necrosis factor (TNF) blocking agents, seems to play an important role in non-response to treatment with these drugs.
To assess the ...relation of clinical response of ankylosing spondylitis (AS) to etanercept with etanercept levels, and the presence of antibodies to etanercept.
Patients with AS were treated with etanercept 25 mg twice weekly, according to the international Assessment in Ankylosing Spondylitis (ASAS) working group consensus statement. Sera were collected at baseline and after 3 and 6 months of treatment. Clinical response was defined as a 50% improvement or as an absolute improvement of 2 points on a (0-10 scale) Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) score. Functional etanercept levels were measured by a newly developed ELISA, measuring the binding of etanercept to TNF. Antibodies against etanercept were measured with a two-site assay and antigen binding test. Clinical data were used to correlate disease activity with serum etanercept levels.
In all, 53 consecutive patients were included. After 3 months of treatment 40 patients (76%) fulfilled the response criteria. Mean etanercept levels were 2.7 mg/litre and 3.0 mg/litre after 3 and 6 months respectively. Characteristics and etanercept levels of responders and non-responders were similar. No antibodies to etanercept were detected with any of the assays.
Etanercept levels of responders and non-responders were similar and no antibodies to etanercept were detected with any of the assays. This study indicates that etanercept is much less immunogenic compared with the other TNF-blocking agents.
Objectives. To analyse whether the beneficial effects of methotrexate in rheumatoid arthritis (RA) could be due to inhibition of inflammatory cytokine production. Methods. Cytokine production was ...studied using whole blood (WB) and mononuclear cells (MNC) of healthy volunteers and RA patients. Cultures were stimulated with either bacterial products such as lipo-oligosaccharide (LOS) or Staphylococcus aureus Cowan I (SAC) to activate monocytes or with monoclonal antibodies to CD3 and CD28 to induce polyclonal T-cell activation. We analysed the effect of methotrexate on cytokine production in these systems. Results. We showed that methotrexate inhibits production of cytokines induced by T-cell activation. Among the cytokines inhibited were interleukin 4 (IL-4), IL-13, IFNγ, tumour necrosis factor-α (TNFα) and granulocyte–macrophage colony-stimulating factor. Inhibition was seen at concentrations easily achieved in plasma of RA patients taking the drug. IL-8 production was hardly influenced by methotrexate. Furthermore, inhibition was dependent on the stimulus; IL-6, IL-8, IL-1β and TNFα production induced by LOS or SAC was only slightly decreased by methotrexate. The addition of folinic acid or thymidine and hypoxanthine reversed the inhibitory effects of methotrexate on cytokine production. Concentrations of methotrexate required for inhibition varied between donors. Oral intake of 10 mg methotrexate by RA patients led to marked inhibition of cytokine production in blood drawn after 2 h. Conclusions. Methotrexate turns out to be an efficient inhibitor of cytokine production induced by T-cell activation in freshly drawn blood. This is due to inhibition of the de novo synthesis of purines and pyrimidines. Cytokines produced by monocytes are hardly affected by methotrexate.
Human serum amyloid P component (SAP) is a glycoprotein structurally belonging to the pentraxin family of proteins, which has a characteristic pentameric organization. Mice with a targeted deletion ...of the SAP gene develop antinuclear Abs, which was interpreted as evidence for a role of SAP in controlling the degradation of chromatin. However, in vitro SAP also can bind to phosphatidylethanolamine, a phospholipid which in normal cells is located mainly in the inner leaflet of the cell membrane, to be translocated to the outer leaflet of the cell membrane during a membrane flip-flop. We hypothesized that SAP, because of its specificity for phosphatidylethanolamine, may bind to apoptotic cells independent of its nuclear binding. Calcium-dependent binding of SAP to early, nonpermeable apoptotic Jurkat, SKW, and Raji cells was indeed observed. Experiments with flip-flopped erythrocytes confirmed that SAP bound to early apoptotic cells via exposed phosphatidylethanolamine. Binding of SAP was stronger to late, permeable apoptotic cells. Experiments with enucleated neutrophils, with DNase/RNase treatment of late apoptotic Jurkat cells, and competition experiments with histones suggested that binding of SAP to late apoptotic cells was largely independent of chromatin. Confocal laser microscopic studies indeed suggested that SAP bound to these apoptotic cells mainly via the blebs. Thus, this study shows that SAP binds to apoptotic cells already at an early stage, which raises the possibility that SAP is involved in dealing with apoptotic cells in vivo.
Objective: To investigate the kinetics of nucleosome leakage from apoptotic cells in an in vitro system and extrapolate the results to autoimmune disease, in particular systemic lupus erythematosus. ...Methods: A sensitive nucleosome enzyme linked immunosorbent assay (ELISA) was developed, using a monoclonal antibody (mAb) against histone 3 and an mAb against nucleosomes. Nucleosome release during apoptotic cell death was studied in Jurkat cells. AnnexinV binding (early apoptosis) and propidium iodide positivity (late apoptosis) of the cells were compared with nucleosome release at different times after apoptosis induction. Results: Nucleosomes appeared in culture supernatant of Jurkat cells 24 to 48 hours after apoptosis induction, when the cells had been late apoptotic for more than 12 hours. Conclusion: Nucleosomes are released from late apoptotic Jurkat cells, with a 12 hour delay from the appearance of AnnexinV binding cells. This result suggests that in vivo scavenger mechanisms have 12 hours to remove apoptotic material from the circulation.
Background: Factor VII‐activating protease (FSAP) is a serine protease that circulates in plasma in its inactive single‐chain form and can be activated upon contact with dead cells. When activated ...by apoptotic cells, FSAP leads to the release of nucleosomes. The serpins C1‐inhibitor and α2‐antiplasmin are reported to be the major inhibitors of FSAP. However, regulation of FSAP activity by Kunitz‐type inhibitors is not well studied.
Objectives: To compare the inhibition of FSAP activity and FSAP‐induced nucleosome release from apoptotic cells by tissue factor pathway inhibitor (TFPI) with that of C1‐inhibitor and α2‐antiplasmin.
Methods: Apoptotic cells were incubated with plasma or FSAP in presence of the inhibitor, and nucleosome release was analyzed with flow cytometry. Monoclonal antibodies against TFPI and altered forms of TFPI were used to investigate which domains of TFPI contribute to FSAP inhibition.
Results and Conclusions: We show that TFPI abrogates FSAP activity and nucleosome release from apoptotic cells. TFPI is a much more efficient inhibitor than C1‐inhibitor or α2‐antiplasmin. The active site of K2 is required for inhibition of FSAP. A direct binding interaction between FSAP and the C‐terminal domain of TFPI is also required for efficient inhibition. Inhibition of FSAP‐induced nucleosome release by recombinant TFPI might, in part, explain the anti‐inflammatory effects of recombinant TFPI infusion observed in animal and human sepsis.
Departments of 1 Pathology, 2 Cardiology, 3 Physiology, and 4 Clinical Chemistry, Vrije Universiteit Medical Center, Amsterdam, The Netherlands; 5 Institute for Cardiovascular Research, Vrije ...Universiteit, Amsterdam, The Netherlands; and 6 Sanquin Research, Central Laboratory for Blood Transfusion, Department of Immunopathology and Laboratory of Experimental and Clinical Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
Submitted 11 April 2005
; accepted in final form 29 June 2005
We examined the relationship between clusterin and activated complement in human heart infarction and evaluated the effect of this protein on ischemic rat neonatal cardiomyoblasts (H9c2) and isolated adult ventricular rat cardiomyocytes as in vitro models of acute myocardial infarction. Clusterin protects cells by inhibiting complement and colocalizes with complement on jeopardized human cardiomyocytes after infarction. The distribution of clusterin and complement factor C3d was evaluated in the infarcted human heart. We also analyzed the protein expression of clusterin in ischemic H9c2 cells. The binding of endogenous and purified human clusterin on H9c2 cells was analyzed by flow cytometry. Furthermore, the effect of clusterin on the viability of ischemically challenged H9c2 cells and isolated adult ventricular rat cardiomyocytes was analyzed. In human myocardial infarcts, clusterin was found on scattered, morphologically viable cardiomyocytes within the infarcted area that were negative for complement. In H9c2 cells, clusterin was rapidly expressed after ischemia. Its expression was reduced after reperfusion. Clusterin bound to single annexin V-positive or annexin V and propidium iodide-positive H9c2 cells. Clusterin inhibited ischemia-induced death in H9c2 cells as well as in isolated adult ventricular rat cardiomyocytes in the absence of complement. We conclude that ischemia induces the upregulation of clusterin in ischemically challenged, but viable, cardiomyocytes. Our data suggest that clusterin protects cardiomyocytes against ischemic cell death via a complement-independent pathway.
ischemia; acute myocardial infarction; membrane flip-flop
Address for reprint requests and other correspondence: P. A. J. Krijnen, VU Univ. Medical Center, Dept. of Pathology, De Boelelaan 1117, 1007 MB Amsterdam, The Netherlands (e-mail: paj.krijnen{at}vumc.nl )
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