The neutralizing effect of heparin on blood-derived antimicrobial compounds: impact on antibacterial activity and inflammatory response.

Acting through a combination of direct and indirect pathogen clearance mechanisms, blood-derived antimicrobial compounds (AMCs) play a pivotal role in innate immunity, safeguarding the host against invading microorganisms. Besides their antimicrobial activity, some AMCs can neutralize endotoxins, preventing their interaction with immune cells and avoiding an excessive inflammatory response. In this study, we aimed to investigate the influence of unfractionated heparin, a polyanionic drug clinically used as anticoagulant, on the endotoxin-neutralizing and antibacterial activity of blood-derived AMCs. Serum samples from healthy donors were pre-incubated with increasing concentrations of heparin for different time periods and tested against pathogenic bacteria (Acinetobacter baumannii, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus) and endotoxins from E. coli, K. pneumoniae, and P. aeruginosa. Heparin dose-dependently decreased the activity of blood-derived AMCs. Consequently, pre-incubation with heparin led to increased activity of LPS and higher values of the pro-inflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6). Accordingly, higher concentrations of A. baumannii, E. coli, K. pneumoniae, and P. aeruginosa were observed as well. These findings underscore the neutralizing effect of unfractionated heparin on blood-derived AMCs in vitro and may lead to alternative affinity techniques for isolating and characterizing novel AMCs with the potential for clinical translation.

Heparin enables the reliable detection of endotoxin in human serum samples using the Limulus amebocyte lysate assay.

The determination of lipopolysaccharide (endotoxin) in serum or plasma samples using Limulus amebocyte lysate (LAL)-based assays is currently not sufficiently reliable in clinical diagnostics due to numerous interfering factors that strongly reduce the recovery of LPS in clinical samples. The specific plasma components responsible for the endotoxin neutralizing capacity of human blood remain to be identified. There are indications that certain endotoxin-neutralizing proteins or peptides, which are part of the host defense peptides/proteins of the innate immune system may be responsible for this effect. Based on our finding that several antimicrobial peptides can be neutralized by the polyanion heparin, we developed a heparin-containing diluent for serum and plasma samples, which enables reliable quantification of LPS measurement in clinical samples using the LAL assay. In a preclinical study involving 40 donors, this improved protocol yielded an over eightfold increase in LPS recovery in serum samples, as compared to the standard protocol. This modified protocol of sample pretreatment could make LPS measurement a valuable tool in medical diagnostics.

Cytokine Removal in Extracorporeal Blood Purification: An in vitro Study.

BACKGROUND/AIMS: Cytokines are among the main target substances that have to be removed effectively in order to improve the patient's health status in the treatment of sepsis, septic shock, and liver diseases. Although there are various medical devices commercially available, the success of their clinical use is limited. The aim of this in vitro study was to compare 3 different medical devices with respect to their clearance for the cytokines interleukin-6 (IL-6), IL-8, IL-1ß, and tumor necrosis factor alpha. The medical devices that were tested are the whole blood adsorbent CytoSorb, the high cutoff filter EMiC2, and the hemofilter HemofeelCH 1.8. METHODS: The study was carried out on the multiFiltrate machine with 1 L human plasma for 8 h. Samples for cytokine quantification were taken at defined time points from the plasma pool. Each experiment was conducted in triplicates, and clearance was calculated for all tested cytokines. RESULTS: All 3 medical devices showed good cytokine removal. The highest clearance for all cytokines was achieved by hemoperfusion with Cytosorb. IL-8 and IL-6 clearance were higher with Hemofeel (continuous venovenous hemodiafiltration) than with EMiC2 (continuous venovenous hemodialysis) because the polymethyl methacrylate (PMMA)-based membrane Hemofeel is able to remove these 2 cytokines by adsorption. Protein and albumin loss was highest withCytosorb and lowest with EMiC2. CONCLUSION: The mechanisms of cytokine removal by blood purification include convection, diffusion, and adsorption. PMMA-based filters are able to combine all 3 mechanisms for certain cytokines. Cytosorb showed the best adsorption kinetics, while dialysis with polystyrene-based membranes offers the best biocompatibility because they do not show any unspecific adsorption of other plasma components.