Comparison between a new multiplex electrochemiluminescence assay and the WHO reference enzyme-linked immunosorbent assay to measure serum antibodies against pneumococcal serotype-specific polysaccharides

BACKGROUND: Two electrochemiluminescence (ECL) assays were developed which, together, can simultaneously measure serum antibodies against pneumococcal capsular polysaccharides (PnPS) for 17 serotypes. The assays were validated for the 13 PnPS included in the 13-valent pneumococcal conjugate vaccine (PCV13). As recommended by the World Health Organization (WHO), we compared the ECL assays with the WHO reference enzyme-linked immunosorbent assay (ELISA) and derived a threshold corresponding to the 0.35 µg/mL threshold established for the WHO reference ELISA for the non-inferiority comparison and licensure of new PCVs against invasive pneumococcal disease. METHODS: A panel of 452 serum samples from children vaccinated with one of the three licensed PCVs was assessed with the ECL assays and the WHO reference ELISA. The ECL assay threshold for the aggregated seven PnPS included in the 7-valent PCV (PCV7) and serotype-specific thresholds were determined using a receiver operating characteristics (ROC) curve-based approach and Deming regression. To evaluate concordance between the ECL assays and the WHO reference ELISA, serostatus agreement rates between both assays and geometric means of the ratios (GMRs) of concentrations obtained with both assays were calculated. RESULTS: The thresholds for the seven aggregated PCV7 serotypes obtained with the ROC curve-based approach and Deming regression approximated 0.35 µg/mL (0.38 and 0.34 µg/mL, respectively). Individual thresholds for the PCV13 serotypes ranged between 0.24 and 0.51 µg/mL across both approaches. Serostatus agreement rates using a 0.35 µg/mL threshold for both assays were ≥86.9% for all PCV13 serotypes. GMRs ranged between 0.85 and 1.25 for 11/13 serotypes and were <1.29 for the two remaining serotypes. CONCLUSION: The ECL assays were comparable to the WHO reference ELISA and offer a sensitive, time- and serum volume-saving method to quantify serotype-specific anti-PnPS antibodies in pediatric sera. A 0.35 µg/mL threshold will be used for each PCV13 serotype to assess PCV immunogenicity in clinical trials

Differential regulation of NF-κB activation and function by topoisomerase II inhibitors

BACKGROUND: While many common chemotherapeutic drugs and other inducers of DNA-damage result in both NF-κB nuclear translocation and DNA-binding, we have previously observed that, depending on the precise stimulus, there is great diversity of the function of NF-κB. In particular, we found that treatment of U-2 OS osteosarcoma cells with the anthracycine daunorubicin or with ultraviolet (UV-C) light resulted in a form of NF-κB that repressed rather than induced NF-κB reporter plasmids and the expression of specific anti-apoptotic genes. Anthracyclines such as daunorubicin can induce DNA-damage though inhibiting topoisomerase II, intercalating with DNA and undergoing redox cycling to produce oxygen free radicals. In this study we have investigated other anthracyclines, doxorubicin and aclarubicin, as well as the anthracenedione mitoxantrone together with the topoisomerase II inhibitor ICRF-193, which all possess differing characteristics, to determine which of these features is specifically required to induce both NF-κB DNA-binding and transcriptional repression in U-2 OS cells. RESULTS: The use of mitoxantrone, which does not undergo redox cycling, and the reducing agent epigallocatechingallate (EGCG) demonstrated that oxygen free radical production is not required for induction of NF-κB DNA-binding and transcriptional repression by these agents and UV-C. In addition, the use of aclarubicin, which does not directly inhibit topoisomerase II and ICRF-193, which inhibits topoisomerase II but does not intercalate into DNA, demonstrated that topoisomerase II inhibition is not sufficient to induce the repressor form of NF-κB. CONCLUSION: Induction of NF-κB DNA-binding and transcriptional repression by topoisomerase II inhibitors was found to correlate with an ability to intercalate into DNA. Although data from our and other laboratories indicates that topoisomerase II inhibition and oxygen free radicals do regulate NF-κB, they are not required for the particular ability of NF-κB to repress rather than activate transcription. Together with our previous data, these results demonstrate that the nature of the NF-κB response is context dependent. In a clinical setting such effects could profoundly influence the response to chemotherapy and suggest that new methods of analyzing NF-κB function could have both diagnostic and prognostic value

Cell Survival from Chemotherapy Depends on NF-κB Transcriptional Up-Regulation of Coenzyme Q Biosynthesis

9 pages and 6 figures.[Background] Coenzyme Q (CoQ) is a lipophilic antioxidant that is synthesized by a mitochondrial complex integrated by at least ten nuclear encoded COQ gene products. CoQ increases cell survival under different stress conditions, including mitochondrial DNA (mtDNA) depletion and treatment with cancer drugs such as camptothecin (CPT). We have previously demonstrated that CPT induces CoQ biosynthesis in mammal cells.[Methodology/Principal Findings] CPT activates NF-κB that binds specifically to two κB binding sites present in the 5′-flanking region of the COQ7 gene. This binding is functional and induces both the COQ7 expression and CoQ biosynthesis. The inhibition of NF-κB activation increases cell death and decreases both, CoQ levels and COQ7 expression induced by CPT. In addition, using a cell line expressing very low of NF-κB, we demonstrate that CPT was incapable of enhancing enhance both CoQ biosynthesis and COQ7 expression in these cells.[Conclusions/Significance] We demonstrate here, for the first time, that a transcriptional mechanism mediated by NF-κB regulates CoQ biosynthesis. This finding contributes new data for the understanding of the regulation of the CoQ biosynthesis pathway.This work was supported by spanish Ministerio de Educacion y Ciencia Grant BFU2005-03017.Peer reviewe

Iron Behaving Badly: Inappropriate Iron Chelation as a Major Contributor to the Aetiology of Vascular and Other Progressive Inflammatory and Degenerative Diseases

The production of peroxide and superoxide is an inevitable consequence of aerobic metabolism, and while these particular "reactive oxygen species" (ROSs) can exhibit a number of biological effects, they are not of themselves excessively reactive and thus they are not especially damaging at physiological concentrations. However, their reactions with poorly liganded iron species can lead to the catalytic production of the very reactive and dangerous hydroxyl radical, which is exceptionally damaging, and a major cause of chronic inflammation. We review the considerable and wide-ranging evidence for the involvement of this combination of (su)peroxide and poorly liganded iron in a large number of physiological and indeed pathological processes and inflammatory disorders, especially those involving the progressive degradation of cellular and organismal performance. These diseases share a great many similarities and thus might be considered to have a common cause (i.e. iron-catalysed free radical and especially hydroxyl radical generation). The studies reviewed include those focused on a series of cardiovascular, metabolic and neurological diseases, where iron can be found at the sites of plaques and lesions, as well as studies showing the significance of iron to aging and longevity. The effective chelation of iron by natural or synthetic ligands is thus of major physiological (and potentially therapeutic) importance. As systems properties, we need to recognise that physiological observables have multiple molecular causes, and studying them in isolation leads to inconsistent patterns of apparent causality when it is the simultaneous combination of multiple factors that is responsible. This explains, for instance, the decidedly mixed effects of antioxidants that have been observed, etc...Comment: 159 pages, including 9 Figs and 2184 reference