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.
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.
► Incidence of antibodies to adalimumab as reported in different studies varies considerably. ► We compared two antigen binding tests and a bridging ELISA. ► Differential susceptibility to drug interference explains most variation between assays. ► Furthermore, the bridging ELISA detects only bivalent drug-specific IgG4. ► The bridging ELISA measures anti-adalimumab only in absence of detectable drug levels.
Antibodies play a central role in immunity by forming an interface with the innate immune system and, typically, mediate proinflammatory activity. We describe a novel posttranslational modification ...that leads to anti-inflammatory activity of antibodies of immunoglobulin G, isotype 4 (IgG4). IgG4 antibodies are dynamic molecules that exchange Fab arms by swapping a heavy chain and attached light chain (half-molecule) with a heavy-light chain pair from another molecule, which results in bispecific antibodies. Mutagenesis studies revealed that the third constant domain is critical for this activity. The impact of IgG4 Fab arm exchange was confirmed in vivo in a rhesus monkey model with experimental autoimmune myasthenia gravis. IgG4 Fab arm exchange is suggested to be an important biological mechanism that provides the basis for the anti-inflammatory activity attributed to IgG4 antibodies.
Objective
Removal of dead cells is essential in the maintenance of tissue homeostasis, and efficient removal prevents exposure of intracellular content to the immune system, which could lead to ...autoimmunity. The plasma protease factor VII–activating protease (FSAP) can release nucleosomes from late apoptotic cells. FSAP circulates as an inactive single‐chain protein, which is activated upon contact with either apoptotic cells or necrotic cells. The purpose of this study was to investigate the role of FSAP in the release of nucleosomes from necrotic cells.
Methods
Necrotic Jurkat cells were incubated with serum, purified 2‐chain FSAP, and/or DNase I. Nucleosome release was analyzed by flow cytometry, and agarose gel electrophoresis was performed to detect DNA breakdown.
Results
Incubation with serum released nucleosomes from necrotic cells. Incubation with FSAP‐deficient serum or serum in which FSAP was inhibited by a blocking antibody was unable to release nucleosomes from necrotic cells, confirming that FSAP is indeed the essential serum factor in this process. Together with serum DNase I, FSAP induced the release of DNA from the cells, the appearance of nucleosomes in the supernatant, and the fragmentation of chromatin into eventually mononucleosomes.
Conclusion
FSAP and DNase I are the essential serum factors that cooperate in necrotic cell DNA degradation and nucleosome release. We propose that this mechanism may be important in the removal of potential autoantigens.
PURPOSE OF REVIEWIn the last decade, biologicals revolutionized rheumatology. An increasing number of patients benefit from biotherapeuticals. However, some patients do not respond to treatment and ...others lose their response after a certain time. Immunogenicity is one of the factors linked to secondary nonresponse but its clinical significance has remained controversial.
RECENT FINDINGSIn recent years, knowledge of how to assess immunogenicity of biologicals has improved. Various reports show an inverse relationship between drug levels and antibody formation against the drug. Studies associated immunogenicity of therapeutic antibodies with clinically significant nonresponse in a subgroup of patients. Clinically relevant immunogenicity is influenced by several factors including dosing and concomitant medication. It has been shown that immunogenicity against biologicals can be persistent or transient.
SUMMARYImmunogenicity affects a significant number of patients treated with biologicals. Monitoring of drug levels as well as of antibodies against therapeutic antibodies may lead to more rational treatment strategies.
Rheumatoid factors are antibodies directed against IgG that may confound immunogenicity testing for therapeutic monoclonal antibodies. We developed antigen-binding assays to monitor ...anti-drug-antibody (ADA) responses against infliximab and adalimumab using F(ab')2 fragments of the drug. This avoids possible detection of rheumatoid factor activity. During development of these assays, a number of sera from patients before treatment as well as several healthy control sera were tested positive. None of these sera contained antibodies specific for the therapeutic mAb. Instead, they were found to contain anti-hinge antibodies. We demonstrate that this aspecific antibody binding can be inhibited by adding F(ab')2 of intravenous immunoglobulin (IVIG), which consists of pooled polyclonal IgG derived from plasma. Using this protocol, anti-infliximab antibodies can be measured specifically without interference by anti-hinge antibodies.
Cell death is a central event in the pathogenesis of sepsis and is reflected by circulating nucleosomes. Circulating nucleosomes were suggested to play an important role in inflammation and were ...demonstrated to correlate with severity and outcome in sepsis patients. We recently showed that plasma can release nucleosomes from late apoptotic cells. Factor VII-activating protease (FSAP) was identified to be the plasma serine protease responsible for nucleosome release. The aim of this study was to investigate FSAP activation in patients suffering from various inflammatory diseases of increasing severity.
We developed ELISAs to measure FSAP-C1-inhibitor and FSAP-α2-antiplasmin complexes in plasma. FSAP-inhibitor complexes were measured in the plasma of 20 adult patients undergoing transhiatal esophagectomy, 32 adult patients suffering from severe sepsis and 8 from septic shock and 38 children suffering from meningococcal sepsis.
We demonstrate plasma FSAP to be activated upon contact with apoptotic and necrotic cells by an assay detecting complexes between FSAP and its target serpins α2-antiplasmin and C1-inhibitor, respectively. By means of that assay we demonstrate FSAP activation in post-surgery patients, patients suffering from severe sepsis, septic shock and meningococcal sepsis. Levels of FSAP-inhibitor complexes correlate with nucleosome levels and correlate with severity and mortality in these patients.
These results suggest FSAP activation to be a sensor for cell death in the circulation and that FSAP activation in sepsis might be involved in nucleosome release, thereby contributing to lethality.
Plasma proteins such as early complement components and IgM are involved in the removal of late apoptotic or secondary necrotic (sn) cells. We have recently described how a plasma protease that could ...be inhibited by the protease inhibitor aprotinin was essential to remove nucleosomes from sn cells. An obvious candidate, plasmin, did indeed have nucleosome-releasing factor (NRF) activity. However, recalcified plasma (r-plasma) retained its NRF activity after plasminogen depletion, which suggests the existence of another protease responsible for NRF activity in plasma. In this study we have used size-exclusion and anion-exchange chromatography to purify the protease responsible for NRF activity in plasma. SDS-PAGE analysis of chromatography fractions containing NRF activity revealed a protein band corresponding with NRF activity. Sequence analysis showed this band to be factor VII-activating protease (FSAP). We developed monoclonal antibodies to FSAP and were able to completely inhibit NRF activity in plasma with monoclonal antibodies to FSAP. Using affinity chromatography we were able to purify single-chain (sc) FSAP from r-plasma. Purified scFSAP efficiently removes nucleosomes from sn cells. We report that factor VII-activating protease may function in cellular homeostasis by catalyzing the release of nucleosomes from secondary necrotic cells.--Zeerleder, S., Zwart, B., te Velthuis, H., Stephan, F., Manoe, R., Rensink, I., Aarden, L. A. Nucleosome-releasing factor: a new role for factor VII-activating protease (FSAP).
Human immunoglobulin G4 (IgG4) is a poor trigger of effector functions and, therefore, is the preferred subclass for therapeutic monoclonal antibodies that merely aim to block their in vivo targets. ...An example is natalizumab, a recombinant IgG4 antibody directed against α4-integrin and used for treatment of multiple sclerosis. Efficient treatment requires that the pharmacokinetics of therapeutic monoclonal antibodies can be accurately monitored. For natalizumab, this requires special precautions due to recently reported structural peculiarities of human IgG4. Here we describe the development of an assay to determine serum levels of natalizumab. Compared with other IgG subclasses, human IgG4 possesses unique structural properties that influence its interactions in both in vivo and in vitro settings. Thus, IgG4 undergoes Fab arm exchange in vivo, resulting in effectively monovalent antibodies. Furthermore, IgG4 is able to bind to other human and nonhuman IgG via Fc interactions. We demonstrate how these features can interfere with measurement of specific IgG4 and describe how we addressed these issues, resulting in an assay that is not sensitive to Fab arm exchange by natalizumab or to IgG4 Fc interactions.
Objectives: Correlation of serum trough infliximab levels and antibodies to infliximab (anti-infliximab) with clinical response in ankylosing spondylitis. Methods: In accordance with the ...international ASsessment in Ankylosing Spondylitis (ASAS) consensus statement, patients were treated with infliximab (5 mg/kg) every 6 weeks after a starting regimen. Preinfusion sera were collected at baseline, 24 and 54 weeks. At every visit, the 20% improvement response (ASAS-20) was assessed and laboratory tests performed. Results: 24 of the 38 (63%) patients fulfilled ASAS-20 response criteria after 24 weeks of treatment and 21 (53%) after 54 weeks. After 54 weeks, 11 (29%) patients showed undetectable serum trough infliximab levels and detectable anti-infliximab; six of these patients developed an infusion reaction. Anti-infliximab was found significantly more often (p = 0.04) in ASAS-20 non-responders compared with responders at week 54. Serum trough infliximab levels were significantly (p<0.0001) lower in patients with (mean 0.02 mg/l) than in those without (12.7 mg/l) anti-infliximab. Conclusions: In ankylosing spondylitis, high levels of serum trough infliximab correlated with a good clinical response. Detection of anti-infliximab within 54 weeks is associated with undetectable serum trough infliximab levels, reduced response to treatment and increased risk of developing an infusion reaction.
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