D-Galactosamine (D-galN) is well established as sensitizing mice and other animals to the lethal effects of TNF, specifically, and by several orders of magnitude. Protection by anti-TNF neutralizing antibody is complete, as is (metabolically-based) protection by uridine. Sensitization occurs regardless of the origin of the released TNF, whether it is released from macrophages and/or T-cells. The same is true for the challenging agent which leads to the release of TNF, whether it is endotoxin, a superantigen, lipoprotein, bacterial DNA, or bacteria, either killed or proliferating. Most studies have utilized endotoxin as the challenging agent, and more than 70 agents have been reported to confer protection against LPS and/or TNF challenge in the model. The model has provided new insight regarding modes of protection, including from dexamethasone, which protects against challenge from LPS but not from challenge by TNF. The D-galN lethality model has also been used to test for synergistic behavior between different bacterial components, and to test for lethality when only small amounts of the challenging agent are available (lipid A chemistry).

Hanovia Type S 200-w lamp through a Pyrex filter until ultraviolet or vpc analysis of the irradiated solution showed maximum product formation (about 5 hr). The retention time of the photo product on a 10-ft vpc column was 6.3 min and for the dienone, 7.8 min. After solvent removal the crude oil obtained was distilled, bp 60°( 0.5 mm), to give 1.5 g (50%) of a crystalline product 18 which on further purification by vpc or sublimation had mp 50-52°; **"° 235, 274, and 320 µ (shoulder) (e 6270, 3240, and 605, respectively); 1690 and 1640 cm-1.

The interactions of lipopolyamines, a class of structurally unique compounds currently being used as transfection (lipofection) agents, with lipopolysaccharide (LPS) have been characterized. Our studies have demonstrated that 1,3-di-oleoyloxy-2-(6-carboxyspermyl)-propylamide), available commercially as DOSPER, binds to purified LPS with an affinity of about 1/10 that of polymyxin B. This essentially nontoxic compound inhibits, in a dose-dependent manner, LPS-induced activation of the Limulus clotting cascade and the production of tumor necrosis factor alpha (TNF-α) interleukin-6 (IL-6), and nitric oxide from LPS-stimulated J774.A1 cells, a murine macrophage-like cell line. Cytokine inhibition is paralleled by decreased steady-state levels of TNF-α and IL-6 mRNA and inhibits the nuclear translocation of nuclear factor kappa B. These findings suggest that the lipopolyamine compound sequesters LPS, thereby blocking downstream cellular activation events that lead to the production of proinflammatory mediators. Administration of DOSPER to d-galactosamine-sensitized mice challenged either with LPS or with Escherichia coliorganisms provided significant protection against lethality both with and without antibiotic chemotherapy. Partial protection is evident in LPS-challenged mice treated with DOSPER as late as 2 to 4 h following the endotoxin challenge. A greater degree of protection is observed in E. coli-challenged animals receiving ceftazidime than in those receiving imipenem, which is probably attributable to the higher levels of LPS released in vivo by the former antibiotic. Potent antiendotoxic activity, low toxicity, and ease of synthesis render the lipopolyamines candidate endotoxin-sequestering agents of potential significant therapeutic value.

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Staphylococcus aureus killed during imipenem or ceftazidime chemotherapy in mice elicited an early release of tumor necrosis factor alpha (TNF-␣) into the systemic circulation. This response was coincident in time with an increase in leukocyte-endothelium adhesive interactions in the microvasculature. Equivalent responses were not observed without the antibiotic treatment (imipenem or ceftazidime). Protective efficacy of the same antibiotic treatment was markedly diminished in D-galactosamine-treated mice compared to controls; e.g., it dropped from 2,000-fold to 70-fold with 4 mg of imipenem per kg given at the time of challenge. Nevertheless, protection was quantitatively restored upon concurrent administration of neutralizing anti-TNF-␣ antibody or 4 mg of dexamethasone per kg to these TNF-␣-hypersensitive mice. Importantly, protection afforded by dexamethasone was not seen when the animals were challenged with viable organisms but without the concurrent administration of antibiotic. An early TNF-␣ response could also be demonstrated upon challenge with Escherichia coli, but in this instance, neither the timing nor the magnitude of that response was influenced by treatment with these antibiotics. We conclude from these studies that the inflammatory response to viable versus killed bacteria may differ markedly depending on the particular bacterium, host sensitivity to TNF-␣, and possibly the Gram stain classification.