1% decomplemented FBS and cytochrome c at a final concentration of 125 g ml. The cells were incubated at 37 C for 5 minutes before the addition of the indicated concentrations of 80 M or 95 M chitosan. Cells were incubated for an additional 10 minutes at 37 C and the reaction stopped on ice for 10 minutes. enzyme inhibitor The samples were then centrifuged at 12,000 g for 2 minutes at 4 C and the opti cal density of the supernatants was read at 540, 550, and 560 nm in a spectrophotometer. The amount of superoxide produced was calculated using the fol lowing formula A550. The absorbance was transformed into the amount of superoxide produced by using a conversion factor of 47. 4, derived from the molar extinction coefficient of cytochrome c.

Release of myeloperoxidase and lactoferrin by polymorphonuclear neutrophils Degranulation was determined using the MPO and lactoferrin assay, as described by Bradley and coworkers and Moc sai and colleagues, respectively. For the MPO assay, PMNs were incubated with 10 mol l cytochalasin B, an actin depolymerizing agent that is known to amplify granule exocytosis, Inhibitors,Modulators,Libraries for 2 minutes at 37 C and Inhibitors,Modulators,Libraries then with the indicated concentrations Inhibitors,Modulators,Libraries of 80 M or 95 M chitosan for 30 minutes at 37 C. A negative control with cyto chalasin B and a positive control with cytochalasin B N formyl methionyl leucyl phenylalanine were also performed. PMNs were then centrifuged for 1 minute at 12,000 g and lysed in HTAB lysis buffer. One hundred microliters of the lysate and cell pellet was mixed with 2. 4 ml of a K2HPO4 buffer before reading the optical density at 460 nm in a spectrophotometer.

Purified human MPO was used to generate Inhibitors,Modulators,Libraries a standard curve. The value % release of MPO corresponds to the ratio of the amount of MPO released the total amount of released and cellular MPO. For the lactoferrin assay, PMNs were incu bated as described above for the MPO experiments. The release of lactoferrin was determined by enzyme linked immu nosorbent assay. Supernatants were diluted 10 fold and 100 fold in 50 mmol l CO2 HCO3 buffer and 100 l of the diluted supernatants or of known concentrations of human lactoferrin Inhibitors,Modulators,Libraries were added to 96 microwell plates and incubated over night at 4 C. Nonspecific binding sites were blocked with phosphate buffered saline supplemented with 0. 5% bovine serum albumin and 0. 5% Tween 20 overnight at room temper ature.

One hundred microliters of rabbit anti human lactoferrin antibody was then added to each well and incubated for 2 hours. One hundred microliters of the secondary antibody, peroxidase conjugated anti rabbit antibody, was then added and incubated for 30 minutes. Each of the above steps was per formed at room U0126 ERK temperature and between each step the plates were repeatedly washed with 1�� Tris buffered saline 0. 1% Tween 20. Tetra methyl benzidene was added before stop ping the reaction with 50 l of 1 mol l sulfuric acid.