ABSTRACT
This technical report addresses the problem of endotoxin contamination of apolipoprotein reagents. Using a bromodeoxyuridine incorporation cell proliferation assay, we observed that human plasma ApoA-I as low as 1 µg/ml resulted in a >90% inhibition in macrophage proliferation. However, not all ApoA-I from different sources showed this effect. We considered the possibility that endotoxin contamination of the apolipoproteins contributed to the differential inhibition of macrophage cell proliferation. Endotoxin alone very potently inhibited macrophage proliferation (0.1 ng/ml inhibited macrophage proliferation>90%). Measurement of endotoxin levels in the apolipoprotein products, including an analysis of free versus total endotoxin, the latter which included endotoxin that was masked due to binding to protein, suggested that free endotoxin mediated inhibition of macrophage proliferation. Despite the use of an advanced endotoxin removal procedure and agents commonly used to inhibit endotoxin action, the potency of endotoxin precluded successful elimination of endotoxin effect. Our findings show that endotoxin contamination can significantly influence apparent apolipoprotein-mediated cell effects (or effects of any other biological products), especially when these products are tested on highly endotoxin-sensitive cells, such as macrophages.
Subject(s)
Apolipoprotein A-I/pharmacology , Cell Proliferation/drug effects , Drug Contamination , Endotoxins/pharmacology , Macrophage Activation/drug effects , Macrophages/drug effects , Apolipoprotein A-I/analysis , Cells, Cultured , Dose-Response Relationship, Drug , Endotoxins/analysis , HumansABSTRACT
Gram-negative bacterial endotoxin is a potent immunostimulant implicated in the development and/or progression of a variety of diseases. The mammalian immune system has both innate and adaptive immune responses to neutralize endotoxin. In this study, a system was developed to monitor bacterial exposure by measuring the extent and nature of endotoxin neutralization in plasma. In control patients, females had higher levels of endotoxin neutralization than males, mirroring clinical outcomes from bacterial infection and sepsis. In addition to the total amount of neutralization, we used inactivation techniques to elucidate the nature of this activity and develop a system to compare early and late immune responses. Using this method to monitor patients with inflammatory bowel disease, we found a more robust total response that relies more on long-term, adaptive components of the immune system and less on early, innate components. Our results indicate that endotoxin neutralization is a valuable method to discern inflammatory bowel disease patients from a control population. Additionally, the nature of neutralization may be valuable in monitoring disease severity and/or the role of medication.
Subject(s)
Biomarkers/metabolism , Endotoxins/metabolism , Inflammatory Bowel Diseases/diagnosis , Neutralization Tests , Acids/pharmacology , Adult , Age Distribution , Aged , Female , Hot Temperature , Humans , Male , Middle Aged , Severity of Illness IndexABSTRACT
Escherichia coli and Salmonella enterica serovar Typhimurium share high degrees of DNA and amino acid identity for 65% of the homologous genes shared by the two genomes. Yet, there are different phenotypes for null mutants in several genes that contribute to DNA condensation and nucleoid formation. The mutant R436-S form of the GyrB protein has a temperature-sensitive phenotype in Salmonella, showing disruption of supercoiling near the terminus and replicon failure at 42 degrees C. But this mutation in E. coli is lethal at the permissive temperature. A unifying hypothesis for why the same mutation in highly conserved homologous genes of different species leads to different physiologies focuses on homeotic supercoil control. During rapid growth in mid-log phase, E. coli generates 15% more negative supercoils in pBR322 DNA than Salmonella. Differences in compaction and torsional strain on chromosomal DNA explain a complex set of single-gene phenotypes and provide insight into how supercoiling may modulate epigenetic effects on chromosome structure and function and on prophage behavior in vivo.