ENDOTOXIN REMOVAL BY POLYMYXIN-B IMMOBILIZED POLYSTYRENE-DERIVATIVE FIBERS DURING IN-VITRO HEMOPERFUSION OF 10-PERCENT HUMAN PLASMA

During gram-negative bacterial sepsis, lipid A, the biologically activ e moiety of endotoxin (ET), activates monocytes and induces the releas e of cytokines. PMX-B, a cationic peptide, binds to lipid A and inhibi ts its activity. Based on this principle, PMX-B was incorporated in po lystyrene-derivative fibers, creating a hemoperfusion column (PMX-20R) that removes ET. After in vitro characterization of the cytokine indu cing 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 (PBM C) 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 millil iters were used to harvest PBMC at baseline, and 10% plasma prepared f rom the rest, was challenged with: 1) 0.01, 1, or 100 ng/ml of purifie d Escherichia coil ET; or 2) 1:1,000 dilution of E. coil, Pseudomonas aeroginosa, 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 m ixed 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 t hree freeze-thaw cycles, and total tumor necrosis factor alpha (TNF al pha) was measured by radioimmunoassay. Before IVH, TNF alpha productio n by PBMC incubated with 10% plasma containing 0.01, 1, or 100 ng/ml o f purified E. coli ET was 1905 +/- 391 pg, 2076 +/- 552 pg, and 5304 /- 1001 pg, respectively. After 2 hours of IVH, the respective decreas e in TNF alpha production was 82 +/- 5% (p = 0.005), 78 +/- 10% (p = 0 .01), and 95 +/- 1% (p = 0.002). Before IVH, TNF alpha production by P BMC incubated with 10% plasma containing 1:1,000 dilution of E. coil, P. aeruginosa or K. pneumoniae was 2896 +/- 273 pg, 1816 +/- 122 pg, a nd 1131 +/- 125 pg, respectively. After 2 hours of IVH, the respective decrease in TNF alpha production was 83 +/- 4% (p < 0.001), 53 +/- 4% (p < 0.001), and 70 +/- 5% (p < 0.001). When IVH was extended to 6 ho urs, the further decrease in TNF alpha production was not statisticall y significant. These results suggest an impressive in vitro removal of ET by PMX-20R from 10% human plasma containing either purified E. coi l FT or E. coil, P. aeruginosa, or K. pneumoniae. Further in vitro stu dies are required, using whole blood challenged with gram-negative bac teria.