David C. Morrison

Binding of polymyxin B to the lipid A portion of bacterial lipopolysaccharides.

Abstract The cationic polypeptide polymyxin B, has been demonstrated to form a stable molecular complex with the lipid A region of bacterial lipopolysaccharides. The interaction between polymyxin B and LPS appears to be stoichiometric, with several lines of evidence suggesting that one molecule of polymyxin B binds one monomer unit of LPS. The formation of LPS-polymyxin B complexes results in the generation of higher mol. wt aggregates with decreased isopynic density.

Two Distinct Mechanisms for the Initiation of Mast Cell Degranulation

Our experiments have provided additional data in support of the concept that different mast cell activators follow distinct biochemical path-ways in the initiation of secretion and degranulation. To do this we have taken advantage of the observation that some serum proteins, and albumins in particular, have the capacity to inhibit selectively the release of amines from rat peritoneal mast cells initiated by some, but not all, stimuli. We show that the relative inhibition of release obtained is independent of the concentration of activator but dependent upon the concentration of albumin, indicating that the inhibitory process does not involve a direct activator--inhibitor interaction. Finally, our data demonstrate that the inhibition does not interfere with the ability of the acitivator to interact with the mast cell. Thus, cells incubated with activator in the presence of inhibitor become increasingly unresponsive, or desensitized, to subsequent challenge with activator in the absence of inhibitor. These combined data therefore provide evidence, that, in addition to a selectivity in the activation/desensitization process initiated by different mast cell stimuli, at least one of the biochemical steps subsequent to the activation step is also not shared by all mast cell stimuli.

The Complement System of the Albacore Tuna, Thunnus Alalunga

Abstract We have examined the hemolytic complement system of one of the higher teleosts, the albacore tuna, Thunnus alalunga . The data suggest that albacore serum possesses natural antibody which crossreacts with sheep erythrocytes and that both classical and alternative pathways contribute to the hemolytic process. Like complement from most other species, albacore complement has a requirement for divalent cations (Mg ++ and Ca ++ ) and activity is thermolabile at 48OC. However, albacore complement was shown to differ from that of other fish in that it was not labile to freezing and thawing. Serum from normal albacore (which are known to thermoregulate) was shown to be efficient over a wide range of temperatures from 11-37 ° C, in contrast to poikilothermic fish which have low temperature optima (4 ° ), or mammals which have high temperature optima (37 ° ).

The use of polymyxin B and C3H/HeJ mouse spleen cells as criteria for endotoxin contamination☆

We have performed experiments designed to evaluate the potential contribution of endotoxin contamination to lymphocyte reponses. Saline and EDTA extracts of 4 different strains of gram negative bacteria were examined for their capacity to initiate mitogenic responses in murine spleen cells. As compared to phenol extracts of these bacteria which contain primarily lipopolysaccharide-LPS, these saline and EDTA extracts were significantly less active in this assay. The mitogenic activity which was present was also manifest in spleen cells from the C3H/HeJ mouse, whereas phenol-extracted LPS preparations were inactive. In addition, mitogenic activity of saline and EDTA extracts was not blocked by polymyxin B, an agent known to abrogate LPS mediated responses. We conclude that LPS contamination may not normally be as significant a problem as had earlier been assumed. However, when endotoxin contamination is present, neither the use of C3H/HeJ spleen cells nor polymyxin B is an appropriate test to evaluate this possibility.

Isolation and characterization of a noncytotoxic mast-cell activator from cobra venom

A protein has been isolated and partially purified from the venom ofNaja naja which causes the noncytolytic release of amines from rat peritoneal mast cells. This protein, termed CVA protein, has been demonstrated to have a molecular weight of 18,500 and sedimentation coefficient of approximately 2s. The activity of CVA protein is destroyed by treatment for 5 min at 100°C, but is not affected at 75°C for 30 min. The ability of CVA protein to initiate mast-cell degranulation has been demonstrated to require both cell energy and calcium. The temperature sensitivity and the cellular requirements distinguish the CVA protein from phospholipase A.

Role of complement in endotoxin initiated lethality in mice

We have examined the role of complement in eliciting a lethal response in C3H/HeJ and C3H/St mice. The results reported here indicate that endotoxin-initiated complement activation, leading to significant drops in circulating C3 levels, is not sufficient to cause lethality. The complement system in both strains was demonstrated to be responsive in vitro to activation both by E. coli 0111:B4 LPS I, an alternative pathway activator in other systems, as well as S. minnesota R595 LPS, which activates almost exclusively the classical pathway. In vivo injection of high (lethal) doses of E. coli 0111:B4 LPS I and S. minnesota R595 LPS causes a significant decrease in the circulating C3 levels of both strains after 4 hr. In contrast, circulating C3 levels were not significantly different from normal values in either strain following injection with minimal (lethal) amounts of E. coli 0111:B4 LPS II, a weakly anticomplementary LPS preparation. In all cases, lethality was observed in only the C3H/St mice, indicating that neither complement activation, nor the lack of it, is responsible for lethality in mice.

Soluble mediators of injury of the microvasculature: Hageman factor and the kinin forming, intrinsic clotting and the fibrinolytic systems

Abstract Studies on Hageman factor have revealed that this protein of approximately 80,000 MW is activated in both solid and fluid phase. In solid phase, the molecule interacts with negatively charged particles without undergoing cleavage. Enzymatic activity is acquired, presumably following a conformational change in the structure of Hageman factor. In fluid phase, the enzymes kallikrein, plasmin, and plasma thromboplastin antecedent (clotting Factor XI) all activated Hageman factor, and in human plasma, the Hageman factor is readily cleaved during this activation. Evidence is presented indicating that kallikrein is the most important fluid phase activator and that the activation with kallikrein is essential for the normal function of the intrinsic clotting, fibrinolytic and kinin forming systems. Information on the role of these systems in immunopathology awaits careful analyses of the function of individual components and means of their accurate detection and quantitation.

Immune responses to hapten-lipopolysaccharide conjugates: 1. Modulation of in vivo antibody responses to the polysaccharide☆☆☆

Abstract The immune responses of mice to various lipopolysaccharides (LPS) and hapten-LPS conjugates were compared. We found that some strains of mice ( A J and BALB/c) produced equivalent amounts of anti-LPS antibody after the injection of either LPS or hapten-LPS conjugates. In contrast, however, other strains of mice (C57BL/6J, C3H/St, DBA/1J, DBA/2J, and Swiss) produced fewer anti-LPS-antibody-secreting cells after stimulation with hapten-LPS conjugates than did mice injected with unsubstituted LPS. The covalent coupling of hapten to LPS changed neither the mitogenic capacity nor the antigenicity of the LPS. The differences in the magnitude of antibody responses to hapten-LPS and LPS in these latter strains of mice occurred in the absence of mature T lymphocytes and was restricted to the primary immune response. Furthermore, these differential responder mice (C57BL/6J) did produce anti-LPS antibody when primed with LPS before challenge with the hapten-LPS conjugate. These data are discussed with respect to both the modulatory capacity of the hapten-LPS in the regulation of the primary immune response to LPS and the biochemical and structural requirements of the hapten-LPS conjugate for immunogenicity.

Anticomplementary activity of lipid A isolated from lipopolysaccharides.

Summary We have examined in detail the relationship between the solubility of the isolated lipid A portion of bacterial lipopoly-saccharides and biological activity. Lipid A isolated by mild acid hydrolysis of the LPS of E. coli 0111:B4, was shown to be an inactivator of complement. This anticomplementary activity was increased over two orders of magnitude after solubilization with triethylamine. In contrast to previously published reports, however, highly active soluble lipid A preparations could be obtained independent of a carrier protein. When such a carrier was present, better than 95% of the carrier did not contribute to the formation of a soluble complex. We conclude that the biological activity of lipid A is highly dependent upon its solubility. We would like to thank Ms. Helen Thomas and Janet Roser for technical assistance. This research is supported by U.S. Public Health Training Grant No. 5TIGM683, International Research Fellowship 1F05TWI1853–01, and by a Fellowship from the Eli Lilly Company (Indianapolis, Indiana).

Two distinct mechanisms for the initiation of mast cell degranulation. II. A specific inhibition of amine release by serum proteins.

Our experiments have provided additional data in support of the concept that different mast cell activators follow distinct biochemical pathways in the initiation of secretion and degranulation. To do this we have taken advantage of the observation that some serum proteins, and albumins in particular, have the capacity to inhibit selectively the release of amines from rat peritoneal mast cells initiated by some, but not all, stimuli. We show that the relative inhibition of release obtained is independent of the concentration of activator but dependent upon the concentration of albumin, indicating that the inhibitory process does not involve a direct activator-inhibitor interaction. Finally, our data demonstrate that the inhibition does not interfere with the ability of the activator to interact with the mast cell. Thus, cells incubated with activator in the presence of inhibitor become increasingly unresponsive, or desensitized, to subsequent challenge with activator in the absence of inhibitor. These combined data therefore provide evidence, that, in addition to a selectivity in the activation/desensitization process initiated by different mast cell stimuli, at least one of the biochemical steps subsequent to the activation step is also not shared by all mast cell stimuli.