Analysis of Inflammatory Mediator Profiles in Sepsis Patients Reveals That Extracellular Histones Are Strongly Elevated in Nonsurvivors.

The timely recognition of sepsis and the prediction of its clinical course are challenging due to the complex molecular mechanisms leading to organ failure and to the heterogeneity of sepsis patients. Treatment strategies relying on a "one-fits-all" approach have failed to reduce mortality, suggesting that therapeutic targets differ between patient subgroups and highlighting the need for accurate analysis of the molecular cascades to assess the highly variable host response. Here, we characterized a panel of 44 inflammatory mediators, including cytokines, chemokines, damage-associated molecular patterns, and coagulation-related factors, as well as markers of endothelial activation in 30 patients suffering from renal failure in the course of sepsis. All patients received continuous veno-venous hemodialysis with either high cut-off filters or with standard filters, and mediators were quantified for all patients at the initiation of dialysis and after 24 h and 48 h. Mediator concentrations in individual patients ranged widely, demonstrating the heterogeneity of sepsis patients. None of the mediators correlated with SAPS III or TISS scores. The overall in-hospital mortality of the study population was 56.7% (57.1% vs. 56.3% for high cut-off vs. standard filter). The two filter groups differed regarding most of the mediator levels at baseline, prohibiting conclusions regarding the effect of standard filters versus high cut-off filters on mediator depletion. The elevation and correlation of damage-associated molecular patterns and markers of endothelial activation gave evidence of severe tissue damage. In particular, extracellular histones were strongly increased and were almost 30-fold higher in nonsurvivors as compared to survivors, indicating their diagnostic and prognostic potential.

Extracellular vesicles are associated with C-reactive protein in sepsis.

There is increasing evidence that C-reactive protein (CRP) can mediate inflammatory reactions following the transformation of functionally inert pentameric CRP (pCRP) into its structural isoform pCRP* and into monomeric CRP (mCRP). This conversion can occur on the membranes of apoptotic or activated cells or on extracellular vesicles (EVs) shed from the cell surface. Here, we characterized the association of CRP with EVs in plasma from sepsis patients using flow cytometry, and found highly elevated levels of total EV counts and CRP+ EVs as compared to healthy individuals. We further assessed the ability of PentraSorb CRP, an extracorporeal device for the adsorption of CRP, to deplete free CRP and CRP+ EVs. Treatment of septic plasma with the adsorbent in vitro resulted in almost complete removal of both, free CRP and CRP+ EVs, while total EV counts remained largely unaffected, indicating the detachment of CRP from the EV surface. EVs from septic plasma elicited a release of interleukin-8 from cultured human monocytes, which was significantly reduced by adsorbent treatment prior to EV isolation. Our findings provide evidence that CRP+ EVs exhibit pro-inflammatory characteristics and can contribute to the spreading of inflammation throughout the circulation on top of their pro-coagulant activity.

Clearance of Selected Plasma Cytokines with Continuous Veno-Venous Hemodialysis Using Ultraflux EMiC2 versus Ultraflux AV1000S.

BACKGROUND: High cutoff hemofilters might support the restoration of immune homeostasis in systemic inflammation by depleting inflammatory mediators from the circulation. METHODS: Interleukin (IL)-6, IL-8, IL-10, and tumor necrosis factor-alpha depletion was assessed in 30 sepsis patients with acute renal failure using continuous veno-venous hemodialysis with high cutoff versus standard filters (CVVHD-HCO vs. CVVHD-STD) over 48 h. RESULTS: The transfer of IL-6 and IL-8 was significantly higher for CVVHD-HCO, as shown by increased IL-6 and IL-8 effluent concentrations. The mean plasma cytokine concentrations decreased over time for all cytokines without detectable differences for the treatment modalities. No transfer of albumin was observed for either of the filters. C-reactive protein remained stable over time and did not differ between CVVHD-HCO and CVVHD-STD, while procalcitonin decreased significantly over 48 h for both treatment modalities. CONCLUSION: CVVHD-HCO achieved enhanced removal of IL-6 and IL-8 as compared to CVVHD-STD, without differentially reducing plasma cytokine levels.

Effect of anticoagulation with citrate versus heparin on the adsorption of coagulation factors to blood purification resins with different charge.

In liver failure, hydrophobic toxins accumulate in the blood circulation. To support hepatic function, extracorporeal blood purification systems have been developed, in which both cationic and neutral adsorbents are used to remove albumin-bound metabolites from blood. An issue of these systems is the additional removal of coagulation factors containing negatively charged γ-carboxyglutamate (Gla) domains, which, in physiological conditions, are shielded by calcium ions. We hypothesized that complexation of calcium ions by citrate leads to exposure of negative Gla domains, resulting in their binding to the positively charged adsorbents. The data presented here confirm that the binding of coagulation factors containing Gla domains to positively charged polymers is enhanced in the presence of citrate as compared to heparin. This effect increased with increasing charge density of the polymer and has important implications for the clinical application of positively charged polymers.

Activation-dependent adsorption of cytokines and toxins related to liver failure to carbon beads.

In the course of severe pathological conditions, such as acute liver failure and sepsis, toxic metabolites and mediators of inflammation are released into the patient's circulation. One option for the supportive treatment of these conditions is plasmapheresis, in which plasma, after being separated from the cellular components of the blood, is cleansed by adsorption of harmful molecules on polymers or activated carbon. In this work, the adsorption characteristics of activated carbon beads with levels of activation ranging from 0 to 86% were assessed for both hydrophobic compounds accumulating in liver failure (bilirubin, cholic acid, phenol and tryptophan) and cytokines (tumor necrosis factor α and interleukin-6). Progressive activation resulted in significant gradual reduction of both bulk density and mean particle size, in an increase in the specific surface area, and to changes in pore size distribution with progressive broadening of micropores. These structural changes went hand in hand with enhanced adsorption of small adsorbates, such as IL-6 and cholic acid and, to a lesser extent, also of large molecules, such as TNF-α.

Neutral styrene divinylbenzene copolymers for adsorption of toxins in liver failure.

In artificial extracorporeal liver support systems, albumin-bound toxins such as bilirubin, bile acids, or aromatic amino acids are removed by adsorption to polymer beads. To overcome the potential weaknesses of anion exchange polymers currently used in liver support, namely, binding of heparin and activation of coagulation, we prepared two series of neutral polystyrene divinylbenzene resins with average pore sizes of 5-6 and 8-9 nm, respectively. In in vitro experiments using human plasma spiked with bilirubin, cholic acid, tryptophan, and phenol, we found that only pores larger than 5-6 nm were accessible to strongly albumin-bound substances, such as bilirubin. On the other hand, less strongly albumin-bound substances, such as bile acids, were efficiently bound by polymers of the small pore size range due to a higher accessible surface area. None of the neutral resins bound significant amounts of heparin. To assess the influence of the polymers on activation of coagulation, generation of thrombin-antithrombin complexes (TAT) was measured at different citrate concentrations. While none of the neutral polymers induced TAT generation, TAT levels were significantly elevated after incubation of plasma with an anion exchange polymer that is in clinical use for extracorporeal liver support. Binding characteristics of the neutral resins for the natural anticoagulants protein C and antithrombin showed remarkable differences, with weak binding of antithrombin but strong removal of protein C, not only for the anion exchanger, but also for neutral polymers of the large pore size range. In conclusion, neutral polystyrene divinylbenzene polymers with a pore size larger than 5-6 nm are efficient adsorbents for albumin-bound toxins that do not induce generation of thrombin-antithrombin complexes.

Efficient adsorption of tumor necrosis factor with an in vitro set-up of the microspheres-based detoxification system.

BACKGROUND: The Microsperes-Based Detoxification System (MDS) represents a flexible therapeutic option for various diseases, depending on the specificity of the adsorbents applied. A potential application of the MDS is the supportive therapy of sepsis. METHODS: Microadsorbents for tumor necrosis factor (TNF) were prepared by immobilization of anti-TNF antibodies on cellulose (1-10 microm) and applied in an experimental set-up using a pool of human plasma (1 liter) spiked with TNF (800 pg/ml) and its soluble receptors (1,000 pg/ml each). Removal of TNF was compared using a plasma filter and the albumin-permeable filter Albuflow. RESULTS: Addition of 4 (2) g of adsorbent to the filtrate circuit reduced TNF concentrations in the pool by 80% (64%). Removal rates did not differ significantly for the different filters. TNF adsorption was not influenced by the presence of excess TNF receptors. CONCLUSIONS: Concentrations of mediators can be efficiently modulated with the MDS using small quantities of specific adsorbents.

Development of specific adsorbents for human tumor necrosis factor-alpha: influence of antibody immobilization on performance and biocompatibility.

To develop adsorbents for the specific removal of tumor necrosis factor-alpha (TNF) in extracorporeal blood purification, cellulose microparticles were functionalized either with a monoclonal anti-TNF antibody (mAb) or with recombinant human antibody fragments (Fab). The TNF binding capacity of the adsorbents was determined with in vitro batch experiments using spiked human plasma (spike: 1200 pg TNF/mL; 1 mg particles in 250 muL plasma). Random immobilization of the full-sized monoclonal antibody to periodate-activated cellulose yielded particles with excellent adsorption capacity (258.1 +/- 48.6 pg TNF per mg adsorbent wet weight). No leaching of antibody was detectable, and the adsorbents retained their activity for at least 12 months at 4 degrees C. We found that the conditions used during immobilization of the antibody (pH, nature of the reducing agent) profoundly influenced the biocompatibility of the resulting adsorbents, especially with respect to activation of the complement system. Particles obtained by random immobilization of the monovalent Fab fragments on periodate-activated cellulose using the same conditions as for immobilization of the mAb exhibited only low adsorption capacity (44 +/- 7 pg/mg adsorbent wet weight). Oriented coupling of the Fab fragments on chelate-epoxy cellulose via a C-terminal histidine tag, however, increased the adsorption capacity to 178.3 +/- 8.6 pg TNF/mg adsorbent wet weight. Thus, in the case of small, monovalent ligands, the orientation on the carrier is critical to retain full binding activity.

Pyrogen transfer across high- and low-flux hemodialysis membranes.

The extent to which bacterial products from contaminated dialysate enter a patient's blood depends upon the type and permeability of the hemodialysis membrane in use. This study was performed to assess the transfer of pyrogenic substances across both high- and low-flux membranes (DIAPES, Fresenius Polysulfone, Helixone, Polyamide S). All experiments were carried out in the saline-saline model. The dialysate pool was contaminated either with purified lipopolysaccharide (LPS) (250 and 500 EU/mL) or with sterile bacterial culture filtrates (20 EU/mL), and in vitro dialysis was performed under diffusive and convective conditions. A significant transfer of endotoxin was observed for both low- and high-flux DIAPES challenged with either LPS or with bacterial culture filtrates. Under identical conditions, no transfer of endotoxins was detectable across Fresenius Polysulfone and Helixone upon challenge with purified LPS. With bacterial culture filtrates, endotoxin concentrations for Polyamide S and Fresenius Polysulfone were about 10% and 1%, respectively, of those measured for DIAPES, whereas no transfer of endotoxin was detectable for Helixone. Using an alternative assay (induction of interleukin-1 receptor antagonist, IL-1Ra, in whole blood), only the DIAPES membrane showed the passage of cytokine-inducing substances. Thus, when saline is present in both the blood and dialysate compartments (i.e., the situation during predialysis priming procedures), dialysis membranes differ profoundly with respect to their permeability to endotoxins.