Eculizumab-C5 complexes express a C5a neoepitope in vivo: Consequences for interpretation of patient complement analyses

Contains fulltext : 177337.pdf (Publisher’s version ) (Open Access)The complement system has obtained renewed clinical focus due to increasing number of patients treated with eculizumab, a monoclonal antibody inhibiting cleavage of C5 into C5a and C5b. The FDA approved indications are paroxysmal nocturnal haemoglobinuria and atypical haemolytic uremic syndrome, but many other diseases are candidates for complement inhibition. It has been postulated that eculizumab does not inhibit C5a formation in vivo, in contrast to what would be expected since it blocks C5 cleavage. We recently revealed that this finding was due to a false positive reaction in a C5a assay. In the present study, we identified expression of a neoepitope which was exposed on C5 after binding to eculizumab in vivo. By size exclusion chromatography of patient serum obtained before and after infusion of eculizumab, we document that the neoepitope was exposed in the fractions containing the eculizumab-C5 complexes, being positive in this actual C5a assay and negative in others. Furthermore, we confirmed that it was the eculizumab-C5 complexes that were detected in the C5a assay by adding an anti-IgG4 antibody as detection antibody. Competitive inhibition by anti-C5 antibodies localized the epitope to the C5a moiety of C5. Finally, acidification of C5, known to alter C5 conformation, induced a neoepitope reacting identical to the one we explored, in the C5a assays. These data are important for interpretation of complement analyses in patients treated with eculizumab

Bypassing adverse injection reactions to nanoparticles through shape modification and attachment to erythrocytes

Intravenously injected nanopharmaceuticals, including PEGylated nanoparticles, induce adverse cardiopulmonary reactions in sensitive human subjects, and these reactions are highly reproducible in pigs. Although the underlying mechanisms are poorly understood, roles for both the complement system and reactive macrophages have been implicated. Here, we show the dominance and importance of robust pulmonary intravascular macrophage clearance of nanoparticles in mediating adverse cardiopulmonary distress in pigs irrespective of complement activation. Specifically, we show that delaying particle recognition by macrophages within the first few minutes of injection overcomes adverse reactions in pigs using two independent approaches. First, we changed the particle geometry from a spherical shape (which triggers cardiopulmonary distress) to either rod- or disk-shape morphology. Second, we physically adhered spheres to the surface of erythrocytes. These strategies, which are distinct from commonly leveraged stealth engineering approaches such as nanoparticle surface functionalization with poly(ethylene glycol) and/or immunological modulators, prevent robust macrophage recognition, resulting in the reduction or mitigation of adverse cardiopulmonary distress associated with nanopharmaceutical administration

Stratégiai szövetségek, a vállalati kapcsolati hálók átalakulása és a versenyképesség

Transplantation and autoimmunit

Electroluminescent TCC, C3dg and fB/Bb epitope assays for profiling Complement cascade activation in vitro using an activated Complement serum calibration standard

Journal ArticleCopyright © 2013 Elsevier B.V. All rights reserved.Electroluminescent assays for epitopes on the complement components C3dg, terminal complement complex (TCC) and factor B/Bb (fB/Bb) have been developed with capture and detection antibodies to produce detection limits C3dg=91±9ng/mL, TCC=3±0.1ng/mL and fB=55.7±0.1ng/mL. The assay performance was assessed against a series of zymosan and heat aggregated IgG (HAIgG) in vitro activations of complement using a calibrated activated complement serum (ACS) as calibration standard. The ACS standard was stable within 20% accuracy over a 6-month period with freeze-thaw cycles as required. Differential activation of the complement cascade was observed for TCC showing a pseudo-first order formation half-life of 3.5h after activation with zymosan. The C3dg activation fragment indicates a 10% total activation for both activation agents. The kinetic-epitope analysis for fB indicates that the capture epitope is on the fB/Bb protein fragment which can then become covered by the formation of C3bBb or C3bBbP complexes during the time course of the cascade.University of Exete

Rewarming after hypothermia after cardiac arrest shifts the inflammatory balance.

Item does not contain fulltextOBJECTIVES: The aim of this study was to simultaneously analyze the key components of the cerebral and systemic inflammatory response over time in cardiac arrest patients during mild therapeutic hypothermia and rewarming. DESIGN AND SETTING: Clinical observational study in a tertiary care university hospital. PATIENTS: Ten comatose patients after out-of-hospital cardiac arrest. INTERVENTIONS: All patients were cooled to 32-34 degrees C for 24 hrs. After 24 hrs patients were passively rewarmed to normothermia. MEASUREMENTS AND MAIN RESULTS: On admission and at 3, 6, 12, 24, and 48 hrs blood samples were taken from the arterial and jugular bulb catheter. Proinflammatory and anti-inflammatory cytokines and chemokines (interleukin-1ra, interleukin-1beta, interleukin-6, interleukin-8, interleukin-10, interleukin-18, monocyte chemotactic protein-1, high-mobility group box-1 and tumor necrosis factor-alpha), complement activation products (C4d, Bb, C3a, and terminal complement complex), and the adhesion molecule soluble intercellular adhesion molecule were measured. Mean temperatures at the start of the study and at 12 and 24 hrs were 33.7 +/- 0.9 degrees C, 32.7 +/- 0.92 degrees C, and 34.5 +/- 1.5 degrees C, respectively. Passive rewarming resulted in a temperature of 37.8 +/- 0.5 degrees C at 48 hrs. The proinflammatory cytokine interleukin-6 increased from 12 to 24 hrs and returned to baseline levels after 48 hrs. In contrast, the chemokines interleukin-8 and monocyte chemotactic protein-1 stayed relatively high from the start and during the hypothermia period, decreasing to baseline levels after 48 hrs. The anti-inflammatory cytokines interleukin-10 and interleukin-1ra did not significantly change during mild therapeutic hypothermia and rewarming, although low values of interleukin-10 were observed after rewarming. A significant increase after rewarming was demonstrated on high-mobility group box-1 concentrations in the jugular bulb, whereas soluble intercellular adhesion molecule increased significantly during hypothermia and remained at this level after rewarming. Complement activation was increased on admission and decreased after induction of hypothermia, followed by a secondary increase during rewarming. No significant differences between any of the biomarkers were found between samples from the arterial and jugular bulb catheter. CONCLUSIONS: Complement activation occurs during rewarming from mild therapeutic hypothermia after cardiac arrest. Interleukin-6 increased already from 12 to 24 hrs, concomitantly with a significant increase in the temperature seen during this period of mild therapeutic hypothermia. The optimal rate of rewarming is unknown. Additional clinical studies are needed to determine the optimal rewarming rate and strategy.1 april 201