ABSTRACT
The structural requirements for the immunopotentiating (adjuvant) effect of endotoxin (ET) were investigated. Mild hydrolysis (0.2 N acetic acid at 95 degrees C) was applied to various ET preparations and the lipid rich (Lipid A) and polysaccharide-rich (PS) preparations obtained were tested as adjuvants on three immunogens: sheep red blood cells (SRBC), L-glutamine: L-lysine: L-alanine containing random synthetic polypeptide (GLA-40), and recombinant HIV viral envelope polypeptide (CBre3). It was found that not only the Lipid A precipitates, but under certain hydrolytic conditions the non-toxic PS preparations were also potent adjuvants. The exact conditions of hydrolysis which led to the isolation of immune adjuvant bacterial products were established. These materials were also tested for endotoxicity (Limulus lysate clotting, chick embryo lethality and local Shwartzman skin reactivity), as well as for TNF generating activities. It was found that TNF generation runs parallel with toxicity of the samples, but it does not follow the adjuvant activity of the isolates. Chemical analysis of the preparations indicated that they did not contain residual ET or Lipid A, however, they did not exclude that deacylated and dephosphorylated skeletal remains of ET are among those components in these preparations which have immunomodulatory activity.
Subject(s)
Adjuvants, Immunologic/pharmacology , Endotoxins/pharmacology , Gene Products, env/immunology , HIV/immunology , Recombinant Proteins/immunology , Animals , Endotoxins/chemistry , Erythrocytes/immunology , HIV Envelope Protein gp120/immunology , HIV Envelope Protein gp41/immunology , Immunoglobulin G/analysis , Lipid A/analogs & derivatives , Lipid A/immunology , Mice , Mice, Inbred ICR , Sheep , Tumor Necrosis Factor-alpha/analysisABSTRACT
The effect of alkaline pH and alkaline hydrolysis on the physico-chemical and biological properties of endotoxin (ET) isolated from Serratia marcescens ATCC 13477 by the Biovin procedure was studied. Major emphasis was put on the ion exchange column chromatography and immune adjuvant activity (ADA) of the alkali treated samples. To measure changes in some endotoxicity parameters, Limulus lysate clotting (LAL), chick embryo lethality, Shwartzman skin reactivity and in vitro TNF release were measured. The toxic properties of ET, with the unique exception of the Shwartzman skin reactivity, rapidly diminished during alkaline treatment. As immunogen CBre3, a recombinant HIV glycoprotein which spans the C terminus of gp 120 and the N terminus of gp 41, was used in CD-1 mice, alkali treated and immediately neutralized ET samples (zero time) were inactive as adjuvants, in some cases immunosuppression could be clearly seen. But if the alkaline hydrolysis was continued for 6 h, the ADA became higher than it had been for the starting ET sample. Further alkaline hydrolysis eliminated the ADA of the samples. Both NaOH and propylamine acted similarly on the ET preparation. Reaction kinetic studies of the NaOH detoxification indicated the cleavage of ester bound acyl groups with low binding energy. Chemical analyses of the samples revealed that changes occurred in the fatty acid composition, characterized by a loss of approximately half of the 3-OH myristic acid content.
Subject(s)
Adjuvants, Immunologic/pharmacology , Endotoxins/pharmacology , Animals , Chick Embryo , Endotoxins/analysis , Endotoxins/toxicity , Fatty Acids/analysis , Hydrogen-Ion Concentration , Hydrolysis , Rabbits , Shwartzman Phenomenon , Tumor Necrosis Factor-alpha/biosynthesisABSTRACT
A new, fast and highly reproducible column liquid chromatographic method was elaborated for the analysis and small-scale preparative isolation of endotoxin from Serratia marcescens Bizio (ATCC No. 264). This procedure detects contaminants of such preparations with high sensitivity and it is capable of separating them from endotoxic components. Extensive heterogeneity of both 5% trichloroacetic acid and phenol-water-extracted endotoxin preparations was recorded. Heterogeneity among the endotoxic components of purified preparations could also be detected by this method. Measurements of biological activities, such as Limulus amoebocyte lysate activation, lymphoblastogenesis (mitogenicity) and tumor necrosis factor (TNF) liberation were carried out on the chromatographically separated fractions. During these studies, non-toxic but in vitro TNF-generating components of crude endotoxin extracts were also detected.
Subject(s)
Chromatography, Liquid , Endotoxins/isolation & purification , Serratia marcescens/analysis , Animals , Biological Assay , Endotoxins/pharmacology , Limulus Test , Lymphocyte Activation , Mice , Mice, Inbred ICR , Phenol , Phenols , Trichloroacetic Acid , Tumor Necrosis Factor-alpha/biosynthesisABSTRACT
A highly cytotoxic, lipid-like compound was isolated from a Serratia marcescens strain currently under identification. We have named the compound DCX for its direct cytotoxic activity on various cell types in culture. DCX was purified by preparative thin layer chromatography from chloroform: methanol = 4:1 extracts of whole bacteria, and is chromatographically homogeneous. The effect of DCX on cells is dose, time, and temperature dependent. DCX is particularly toxic to the mastocytoma cell line P815 (TD50 = 75 pg/ml). Three other malignant or transformed murine cell lines were sensitive to the cytotoxic action of DCX. The effect of DCX was also tested on normal cells (human gingival fibroblasts), which showed greater resistance to DCX than the other cells tested.
