Endotoxin removal by polymyxin-B immobilized polystyrene-derivative fibers during in vitro hemoperfusion of 10% human...

During gram-negative bacterial sepsis, lipid A, the biologically active moiety of endotoxin (ET), activates monocytes and induces the release of cytokines. PMX-B, a cationic peptide, binds to lipid A and inhibits its activity. Based on this principle, PMX-B was incorporated in polystyrene-derivative fibers, creating a hemoperfusion column (PMX-20R) that removes ET. After in vitro characterization of the cytokine inducing potency of three gram-negative bacterial challenges, the authors evaluated the in vitro efficacy of PMX-20R in a model using 10% human plasma. Cytokine production by peripheral blood mononuclear cells (PBMC) incubated with plasma before and after in vitro hemoperfusion (IVH) was used as the index of ET removal. One hundred forty milliliters of heparinized blood were obtained from healthy volunteers. Forty milliliters were used to harvest PBMC at baseline, and 10% plasma prepared from the rest, was challenged with: 1) 0.01, 1, or 100 ng/ml of purified Escherichia coli ET; or 2) 1:1,000 dilution of E. coli, Pseudomonas aeruginosa, or Klebsiella pneumoniae. IVH was performed at 100 ml/min at 37 degrees C for up to 6 hours. One half milliliter samples, drawn before and at designated time intervals after the start of IVH, were mixed with a 0.5 ml suspension of 5 x 10(6) PBMC/ml from the same donor, and incubated for 24 hours at 37 degrees C. PBMC were subjected to three freeze-thaw cycles, and total tumor necrosis factor alpha (TNFalpha) was measured by radioimmunoassay. Before IVH, TNFalpha production by PBMC incubated with 10% plasma containing 0.01, 1, or 100 ng/ml of purified E. coli ET was 1905+/-391 pg, 2076+/-552 pg, and 5304+/-1001 pg, respectively. After 2 hours of IVH, the respective decrease in TNFalpha production was 82+/-5% (p = 0.005), 78+/-10% (p = 0.01), and 95+/-1% (p = 0.002). Before IVH, TNFalpha production by PBMC incubated with 10% plasma containing 1:1,000 dilution of E. coli, P. aeruginosa or K. pneumoniae was 2896+/-273 pg, 1816+/-122 pg, and 1131+/-125 pg, respectively. After 2 hours of IVH, the respective decrease in TNFalpha production was 83+/-4% (p < 0.001), 53+/-4% (p < 0.001), and 70+/-5% (p < 0.001). When IVH was extended to 6 hours, the further decrease in TNFalpha production was not statistically significant. These results suggest an impressive in vitro removal of ET by PMX-20R from 10% human plasma containing either purified E. coli ET or E. coli, P. aeruginosa, or K. pneumoniae. Further in vitro studies are required, using whole blood challenged with gram-negative bacteria.