682
CORRESPONDENCE Detoxification of Meningococcal Endotoxin by Polymyxin B
Michael Cooperstock Department ofChild Health. University ofMissouri School ofMedicine, Columbia References
Reprints or correspondence: Dr. Michael Cooperstock, Department of Child Health, University of Missouri School of Medicine, Columbia, MO 65212. The Journal of Infectious Diseases
1992;166:682
© 1992 by The University of Chicago. All rights reserved.
0022-1899/92/6603-0042$01.00
Reply To the Editor-Cooperstock's comments [1] are well taken. Meningococcal lipooligosaccharides (LOS) may differ in the structure of their oligosaccharides [2, 3] as well as in the phosphoryl content and fatty acids of their lipid A moieties [3]. Growth conditions may influence this heterogeneity ofmen ingococcal LOS [2]. The ability of LOS to gelate limulus amebocyte lysate (LAL) or to bind polymyxin B may be influenced by differences in LOS structure. Roth et al. [3] recently reported 500fold variation in the activities of meningococcal LOS in the LAL assay and ascribed this variation to differences in the lipid A structure. Gu and Tsai [4] have also noted that the method ofpurification of LOS may affect its biologic activities. Meningococcal LOS purified by deoxycholate extraction and gel filtration chromatography was 10- to 100-fold more active in the LAL assay and in rabbit pyrogenicity than was LOS purified by the traditional phenol-water method [4]. The greater biologic activity is probably caused by the higher degree ofdeaggregation ofdeoxycholate-treated LOS. We therefore further evaluated the effect of polymyxin B on the ability of various meningococcal LOS and Escherichia coli Iipopolysaccharides (LPS) to activate LAL. Meningococcal LOS from strains 891, 35E, 8532, and 126E purified by the phenol-
Reprints or correspondence: Dr. Gary R. Fleisher, Division of Emergency Medicine, Children's Hospital, 300 Longwood Ave., Boston, MA 02115. The Journal of Infectious Diseases
1992;166:682-3
© 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6603-0043$01.00
I. Baldwin G, Alpert G, Caputo GL, et al. Effect of polymyxin B on experimental shock from meningococcal and Escherichia coli endotoxins. J Infect Dis 1991;164:542-9. 2. Cooperstock MS. Inactivation ofendotoxin by polymyxin B. Antimicrob Agents Chemother 1974;6:422-5.
water method were provided by W. Zollinger and B. Brandt (Walter Reed Army Institute of Research, Washington, DC). LOS from strain M986 purified by deoxycholate extraction and gel filtration chromatography [4] was provided by C. Tsai (Center for Biologics Evaluation and Review, Bethesda, MD). E. coli LPS from strains 0 III b4 and J 5 were obtained from List Laboratories (Campbell, CA), from strain Ol8ac from A. Cross (Walter Reed Army Institute of Research, Washington, DC), and from strain 0113 from Associates of Cape Cod (Woods Hole, MA). The concentration of each endotoxin required to produce 50%gelation ofLAL, defined as the LAL response so(LR so), was measured as previously described [5] with or without polymyxin B at to p.g/mL (table I). The LR so of some LOS preparations showed assay-to-assay variation, and the magnitude of the neutralizing effect of polymyxin B also showed variation. However, the meningococcal LOS consistently showed no neutralization by polymyxin B or only low-level neutralization (less than fivefold), and the E. coli LPS consistently showed higher levels of neutralization. Differences in the LR so of different meningococcal LOS and E. coli LPS were noted. The meningococcal LOS extracted with deoxycholate (M986) was more active in the LAL assay and was neutralized to a greater extent by polymyxin B than the other LOS extracted with phenol water. The current results differ from our previous report in that the LR so ofM986 LOS is lower (0.5 pg/mL vs. 4.0 pg/ml, previously) and the neutralization by polymyxin B at 10 p.g/mL was fourfold compared with none previously [6]. The reason for these differences is not clear but could be related to differences in LAL lots, polymyxin B lots, or the state of aggregation of the M986 LOS. Since Cooperstock [I] noted greater neutralization with me-
Downloaded from http://jid.oxfordjournals.org/ at University of New South Wales on September 13, 2015
To the Editor-Baldwin et al. [I] describe the failure of polymyxin B to detoxify meningococcal endotoxin, either in rabbits or in vitro using the limulus assay. In contrast, Escherichia coli endotoxin was neutralized in both systems. A single preparation of meningococcal endotoxic lipooligosaccharide was used for their experiments. Although I have not studied this issue extensively, I did find, some years ago, that polymyxin effected a sharp reduction in limulus assay activity in the diluted broth culture supernatant of a meningococcal strain isolated from the spinal fluid ofa patient
with meningitis [2]. Activity was decreased 30-fold in the presence of 1.0 p.gof polymyxin/ml, and 10,000-fold with to p.g of polymyxin/rnl., In unpublished experiments, it was also possible to sharply decrease limulus assay activity of diluted spinal fluid from another patient with meningococcal meningitis with the addition of polymyxin directly to the fluid. It seems best to conclude that the interaction of polymyxin with meningococcal endotoxin needs further study in order to better understand these conflicting data.
CORRESPONDENCE Detoxification of Meningococcal Endotoxin by Polymyxin B
Michael Cooperstock Department ofChild Health. University ofMissouri School ofMedicine, Columbia References
Reprints or correspondence: Dr. Michael Cooperstock, Department of Child Health, University of Missouri School of Medicine, Columbia, MO 65212. The Journal of Infectious Diseases
1992;166:682
© 1992 by The University of Chicago. All rights reserved.
0022-1899/92/6603-0042$01.00
Reply To the Editor-Cooperstock's comments [1] are well taken. Meningococcal lipooligosaccharides (LOS) may differ in the structure of their oligosaccharides [2, 3] as well as in the phosphoryl content and fatty acids of their lipid A moieties [3]. Growth conditions may influence this heterogeneity ofmen ingococcal LOS [2]. The ability of LOS to gelate limulus amebocyte lysate (LAL) or to bind polymyxin B may be influenced by differences in LOS structure. Roth et al. [3] recently reported 500fold variation in the activities of meningococcal LOS in the LAL assay and ascribed this variation to differences in the lipid A structure. Gu and Tsai [4] have also noted that the method ofpurification of LOS may affect its biologic activities. Meningococcal LOS purified by deoxycholate extraction and gel filtration chromatography was 10- to 100-fold more active in the LAL assay and in rabbit pyrogenicity than was LOS purified by the traditional phenol-water method [4]. The greater biologic activity is probably caused by the higher degree ofdeaggregation ofdeoxycholate-treated LOS. We therefore further evaluated the effect of polymyxin B on the ability of various meningococcal LOS and Escherichia coli Iipopolysaccharides (LPS) to activate LAL. Meningococcal LOS from strains 891, 35E, 8532, and 126E purified by the phenol-
Reprints or correspondence: Dr. Gary R. Fleisher, Division of Emergency Medicine, Children's Hospital, 300 Longwood Ave., Boston, MA 02115. The Journal of Infectious Diseases
1992;166:682-3
© 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6603-0043$01.00
I. Baldwin G, Alpert G, Caputo GL, et al. Effect of polymyxin B on experimental shock from meningococcal and Escherichia coli endotoxins. J Infect Dis 1991;164:542-9. 2. Cooperstock MS. Inactivation ofendotoxin by polymyxin B. Antimicrob Agents Chemother 1974;6:422-5.
water method were provided by W. Zollinger and B. Brandt (Walter Reed Army Institute of Research, Washington, DC). LOS from strain M986 purified by deoxycholate extraction and gel filtration chromatography [4] was provided by C. Tsai (Center for Biologics Evaluation and Review, Bethesda, MD). E. coli LPS from strains 0 III b4 and J 5 were obtained from List Laboratories (Campbell, CA), from strain Ol8ac from A. Cross (Walter Reed Army Institute of Research, Washington, DC), and from strain 0113 from Associates of Cape Cod (Woods Hole, MA). The concentration of each endotoxin required to produce 50%gelation ofLAL, defined as the LAL response so(LR so), was measured as previously described [5] with or without polymyxin B at to p.g/mL (table I). The LR so of some LOS preparations showed assay-to-assay variation, and the magnitude of the neutralizing effect of polymyxin B also showed variation. However, the meningococcal LOS consistently showed no neutralization by polymyxin B or only low-level neutralization (less than fivefold), and the E. coli LPS consistently showed higher levels of neutralization. Differences in the LR so of different meningococcal LOS and E. coli LPS were noted. The meningococcal LOS extracted with deoxycholate (M986) was more active in the LAL assay and was neutralized to a greater extent by polymyxin B than the other LOS extracted with phenol water. The current results differ from our previous report in that the LR so ofM986 LOS is lower (0.5 pg/mL vs. 4.0 pg/ml, previously) and the neutralization by polymyxin B at 10 p.g/mL was fourfold compared with none previously [6]. The reason for these differences is not clear but could be related to differences in LAL lots, polymyxin B lots, or the state of aggregation of the M986 LOS. Since Cooperstock [I] noted greater neutralization with me-
Downloaded from http://jid.oxfordjournals.org/ at University of New South Wales on September 13, 2015
To the Editor-Baldwin et al. [I] describe the failure of polymyxin B to detoxify meningococcal endotoxin, either in rabbits or in vitro using the limulus assay. In contrast, Escherichia coli endotoxin was neutralized in both systems. A single preparation of meningococcal endotoxic lipooligosaccharide was used for their experiments. Although I have not studied this issue extensively, I did find, some years ago, that polymyxin effected a sharp reduction in limulus assay activity in the diluted broth culture supernatant of a meningococcal strain isolated from the spinal fluid ofa patient
with meningitis [2]. Activity was decreased 30-fold in the presence of 1.0 p.gof polymyxin/ml, and 10,000-fold with to p.g of polymyxin/rnl., In unpublished experiments, it was also possible to sharply decrease limulus assay activity of diluted spinal fluid from another patient with meningococcal meningitis with the addition of polymyxin directly to the fluid. It seems best to conclude that the interaction of polymyxin with meningococcal endotoxin needs further study in order to better understand these conflicting data.
