DETECTION OF BACTERIAL PYROGENS ON THE BASIS OF THEIR EFFECTS ON GAMMA-INTERFERON-MEDIATED FORMATION OF NEOPTERIN OR NITRITE IN CULTURED RVMONOCYTE CELL-LINES

In a number of mammalian cell types, pteridine biosynthesis from guano sine 5'-triphosphate and formation of nitric oxide from L-arginine are induced by gamma interferon (IFN-gamma) and bacterial lipopolysacchar ide (LPS). We assessed the possibility of using such metabolic alterat ions for the in vitro detection of pyrogens. Products from gram-negati ve and gram-positive bacteria and related synthetic compounds were tes ted for their potential to induce either of these pathways. Stimulatio n of pteridine biosynthesis was monitored as the formation of neopteri n in the human myelomonocytic cell line THP-1. The formation of nitric oxide was determined as nitrite in murine J774A.1 macrophage cultures , The substances tested included toxic and detoxified parts of LPS and lipid A from Escherichia coli, Salmonella typhimurium, Salmonella min nesota, and Klebsiella pneumoniae as well as lipoteichoic acid and tox ic shock syndrome toxin 1 from Staphylococcus aureus, Furthermore, two cell wall compounds from Mycobacterium tuberculosis, trehalose 6,6'-d imycolate and N-acetylmuramyl-L-alanyl-D-isoglutamine, which are activ e components of Freund's adjuant, were used. When applied as a single stimulus, only the whole LPS molecule potently stimulated neopterin or nitrite formation, Lipid A and products from gram-positive bacteria w ere weakly active, For neopterin formation, lipid A required the prese nce of fetal calf serum, Besides detoxified LPS and independently from the presence of serum, all bacterial compounds tested strongly increa sed the effects mediated by IFN-gamma. Our results show that bacterial pyrogens can be detected by monitoring the formation of neopterin or nitrite, This may provide a basis for the development of an in vitro a ssay for the detection of pyrogenic contamination with the aim of repl acing the currently used animal test.