Production of TNFα is not due to endotoxin, bacterial DNA, RNA or contaminating proteins in <i>B. anthracis</i> peptidoglycan.

<p>A) The Limulus amebocyte lysate assay was used to test PGN (10 µg/ml), LPS (1 pg/ml, 10 pg/ml, 100 pg/ml) and endotoxin-free water for endotoxin activity, Endotoxin units (EU)/ml. Data are expressed as mean±SEM of 3 replicates. Statistical significance was determined for LPS (10 pg/ml) compared with PGN (10 µg/ml) by unpaired, two-tailed t test; p = 0.49, indicating no significant difference in level of endotoxin activity. (B) PB was stimulated with 10 pg/ml LPS 6 hours and TNFα positive monocytes were measured by flow cytometry. (C) PB was stimulated with PGN or LPS with and without polymixin B (PMB) (10 µg/ml) to bind endotoxin. TNFα production is expressed as fold difference±SEM of MFI of TNFα positive monocytes for 2 separate preparations on two individuals, 2 replicates each. Statistical significance was determined by ANOVA with Bonferroni post test. *, p<0.001 <i>versus</i> 1 mg/ml LPS without PMB. (D) <i>B. anthracis</i> PGN was digested with the indicted hydrolytic enzymes for 24 hours. The preparations were heat inactivated and used to stimulate PB at 10 µg/ml PGN along with an equivalent preparation of untreated PGN. The percent of TNFα positive monocytes was measured by intracellular cytokine staining. Data are expressed as the mean±SEM of three separate preparations. Statistical significance was performed on results from the enzyme treated PGN stimulations <i>versus</i> stimulation with untreated PGN by ANOVA with Bonferroni post test. *, p<0.001 <i>versus</i> untreated PGN.