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Control and elimination of bacterial contaminants in pharmacological experiments Samples undergoing pharmacological screening may be contaminated with bacteria and/or complex molecules of their outer membrane. The most potent signal molecule of Gram-negative bacteria is lipopolysaccharide (LPS; endotoxin). The major biologically active molecules of Gram-positive bacteria are peptidoglycans (PGN) and lipoteichoic acid (LTA). Acting through distinct receptor systems such as membrane receptor CD14, Toll-like receptors TLR4, TLR2 and nucleotide-binding oligomerization domain 2 (NOD2), they activate various signaling pathways and thus possess pleiotropic metabolism and biological effects. Conceivably, the data and interpretation of pharmacological experiments may be seriously biased unless the presence of bacterial contaminants is not ruled out. The control of bacterial contamination of experimental samples is almost exclusively, if at all, focused on LPS. It can be detected using the kinetic chromogenic Limulus amebocyte lysate (LAL) assay. The most frequently used method to remove LPS from samples is based on the capacity of LPS to bind the antibacterial cyclic lipopeptide polymyxin B (PMX). The aims of our experiments were to determine conditions required for a) reliable detection of LPS using the LAL assay, b) safe use and reliable removal of LPS by PMX, and c) to introduce an alternative method of bacterial decontamination based on the cutoff centrifugal microfiltration. Materials and methods. Concentration of LPS was determined by the LAL assay using kinetic-QCL kit (Cambrex Bio Science, Walkersville, MD). If indicated, the samples were incubated with PMX (Sigma-Aldrich) at RT for 2 h. Alternatively the Amicon® Ultra 0.5 mL centrifugal devices (Millipore Corp., Billerica, MA) with cutoff filter membranes of 3, 10, 30, 50, and 100 kDa were used. The decontamination effectiveness of the methods was monitored by the LAL assay and the functional nitric oxide (NO) assay. For this purpose, the rat peritoneal cells (2 x 106/mL) were cultured in the presence of contaminated and decontaminated samples. The supernatant concentration of nitrites was assayed spectrophotometrically (540 nm) at the interval of 24 h of cell culture using Griess reagent. Major findings. 1/ The LAL assay may provide both false negative and false positive results: a) Even high amounts of LPS do not show any response in the presence of fetal bovine serum which is commonly used in culture media; b) The LAL assay is not LPS-specific. The products of Gram-positive bacteria such as LTA and PGN show the positive response as well. 2/ If PMX is used, its concentration reliably removing the LAL positivity as well as the NO-augmenting effects of LPS must be 10,000-fold higher than the concentration of LPS. The use of PMX is restricted to concentrations of ≤25 µg/mL; higher concentrations become cytotoxic. 3/ In contrast to the capability to remove LPS, PMX is inefficient in removing neither the LAL positivity nor NO-stimulatory effects of products of Gram-positive bacteria, LTA and PGN. 4/ The centrifugal microfiltration removes all LPS, LTA and PGN as proved by negative LAL response and complete inhibition of NO production triggered by these bacterial products. The 100 kDa cutoff filters are fully effective. Conclusion. The cutoff microfiltration can reliably remove not only LPS of Gram-negative bacteria but also LTA and PGN of Gram-positive bacteria. It may be suggested as a highly advantageous alternative to hitherto broadly used PMX method, which is selective for LPS solely. Acknowledgements. The research was supported by grants CZ: GA ČR:303/12/0172 and CZ: GA ČR: 303/12/0535 from the Grant Agency of the Czech Republic.
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