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Endotoxin is one of the principal mediators of Gram-negative septic shock. Pretreatment with monophosphoryl lipid A, a hydrolyzed derivative of endotoxin from Salmonella minnesota R595, induces endotoxin tolerance and nonspecific resistance to infection in experimental animals. The present clinical trial was undertaken to test the response to monophosphoryl lipid A in humans and the ability of monophosphoryl lipid A to attenuate the response of normal human volunteers to U.S. Reference Ec-5 endotoxin.Prospective, double-blind, randomized, controlled trial.Clinical research center.Forty-four healthy volunteers.In part 1 of the study, 29 volunteers were randomized in varying ratios to receive vehicle control or monophosphoryl lipid A intravenously in a double-blind dose escalation trial. In part 2 of the study, 12 volunteers were randomized to receive either monophosphoryl lipid A (20 micrograms/kg) or vehicle control and, 24 hrs later, all 12 volunteers were challenged with U.S. Reference Ec-5 endotoxin (20 units/kg intravenous, bolus injection). Systemic response to endotoxin challenge was evaluated and compared between the monophosphoryl lipid A and vehicle control-pretreated subjects.In part 1 of the study, subjective effects and increases in cytokine levels were not observed until a dose of 10 micrograms/kg of monophosphoryl lipid A was administered. Six volunteers receiving a maximum dose of 20 micrograms/kg experienced mild-to-moderate symptoms that did not require therapy. Moderate increases in temperature, heart rate, and tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-8 release were observed. IL-1 alpha and IL-1 beta were not detected but a significant increase in IL-1 receptor antagonist was observed. In part 2 of the study, monophosphoryl lipid A pretreatment reduced the number of volunteers who experienced one or more subjective complaints after endotoxin administration (3/6 vs. 6/6; p = .09). The febrile response and tachycardic response to endotoxin were significantly reduced by pretreatment with monophosphoryl lipid A. Monophosphoryl lipid A-pretreated volunteers demonstrated significantly reduced concentrations of TNF-alpha after endotoxin challenge, as compared with subjects treated with vehicle control (84 +/- 76 vs. 244 +/- 128 pg/mL; p.05). IL-6 concentrations (100 +/- 91 vs. 268 +/- 171 pg/ml; p.05) and IL-8 concentrations (136 +/- 86 vs. 632 +/- 323 pg/mL; p.05) elicited by endotoxin challenge were also significantly reduced by monophosphoryl lipid A pretreatment.Data indicate that monophosphoryl lipid A, in a dose 10,000 times that of endotoxin, used in experimental pyrogenicity trials, is well tolerated in human volunteers. Pretreatment of normal human volunteers with monophosphoryl lipid A attenuated the systemic response to bacterial endotoxin. These data support further clinical testing of monophosphoryl lipid A for the prevention or amelioration of the severe sequelae of sepsis.

The antibody responses of outbred normal mice and nude mice injected with alkaline detoxified lipopolysaccharide (Alk-LPS) were measured. In some cases the antibody responses to Alk-LPS were compared to responses made against lipopolysaccharide (LPS) and native protoplasmic polysaccharide (NPP). The kinetics of the primary responses to Alk-LPS and NPP were similar, whereas LPS stimulated a more rapid appearance of antibodies in the primary responses. Alk-LPS stimulated only primary antibody responses in both types of mice and sensitized nude mice for secondary responses which could be triggered with LPS. However, secondary antibody responses could not be triggered in normal mice primed with Alk-LPS. These data suggested that, on a functional basis, Alk-LPS possessed the specific antigenic signal associated with LPS antigens but lacked the second nonspecific mitogenic signal dependent on the lipid A portion of LPS.

In an attempt to elucidate the nature of the endotoxin-altering reaction, normal sera from animals of different phylogenetic levels were tested for capacity to inactivate endotoxin from Escherichia coli. Inactivation was assayed quantitatively by reduction in capacities of endotoxin to be precipitated by homologous antiserum and to kill mice, and by changes in its immunodiffusion pattern. Sera from species of Mammalia, Aves, Reptilia, Amphibia, Osteichthyes, and Chondrichthyes were shown to have similar capacities to inactivate endotoxin, whereas sera from cyclostomes and hemolymphs from crustaceans altered endotoxin only slightly or not at all. In general, the qualitative nature of the endotoxin-altering reaction by these sera was similar in that the reaction was inhibited by calcium (100 [tmoles/ml) and, excepting carp and turtle sera, it was heat stable (56 C for 30 min). Inactivation was greater when endotoxin was incubated with serum at 37 C rather than at 3 C or 26 C. It is postulated that an endotoxin-altering mechanism similar to that of human serum originated phylogenetically between hagfish and shark.

Functional separation of the two signals involved in stimulating immunological responses was achieved through the judicious use of two natural bacterial antigens. Native protoplasmic polysaccharide (NPP) extracted from Escherichia coli was immunochemically identical to the lipopolysaccharide (LPS) extracted from the same organism. However, NPP was not endotoxic, not mitogenic, did not fix complement, and was immunologically independent of T cells. The NPP, which appeared to contain only the antigenic signal, could induce a primary antibody response in mice and could sensitize mice for a secondary response. However, the antigenic signal contained in NPP was insufficient to trigger a secondary response in mice primed with either NPP or LPS. LPS, containing both the antigenic and second signals, was required to trigger a secondary response in primed mice.

Development in chicken embryos of increased resistance to lethal effects of endo-toxin could not be quantitatively accounted for by increased endotoxin-inactivating capacity of their sera.