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PubMed2004

Aggregates are the biologically active units of endotoxin.

For the elucidation of the very early steps of immune cell activation by endotoxins (lipopolysaccharide, LPS) leading to the production and release of proinflammatory cytokines the question concerning the biologically active unit of endotoxins has to be addressed: are monomeric endotoxin molecules able to activate cells or is the active unit represented by larger endotoxin aggregates? This question has been answered controversially in the past. Inspired by the observation that natural isolates of lipid A, the lipid moiety of LPS harboring its endotoxic principle, from Escherichia coli express a higher endotoxic activity than the same amounts of the synthetic E. coli-like hexaacylated lipid A (compound 506), we looked closer at the chemical composition of natural isolates. We found in these isolates that the largest fraction was hexaacylated, but also significant amounts of penta- and tetraacylated molecules were present that, when administered to human mononuclear cells, may antagonize the induction of cytokines by biologically active hexaacylated endotoxins. We prepared separate aggregates of either compound 506 or 406 (tetraacylated precursor IVa), mixed at different molar ratios, and mixed aggregates containing both compounds in the same ratios. Surprisingly, the latter mixtures showed higher endotoxic activity than that of the pure compound 506 up to an admixture of 20% of compound 406. Similar results were obtained when using various phospholipids instead of compound 406. These observations can only be understood by assuming that the active unit of endotoxins is the aggregate. We further confirmed this result by preparing monomeric lipid A and LPS by a dialysis procedure and found that, at the same concentrations, only the aggregates were biologically active, whereas the monomers showed no activity.

Animals↗
PubMed2000

Preliminary report on the results of the second phase of a round- robin endotoxin assay study using cotton dust.

In an on-going endotoxin assay study, a two-part interlaboratory endotoxin assay study has been completed. The purpose of the study was to compare the variation in assay results between different laboratories, and, if the variation was high, to see if a common protocol would reduce the variation. In both parts of the study, membrane filters laden with the same approximate amount and type of cotton dust were sent for analysis to laboratories that "routinely" perform endotoxin analyses. First, each of these laboratories performed the analysis using the methodology common to its laboratory. In the second part of the study, membrane filters with cotton dust were again sent to the same laboratories where the analyses were performed as before but with a common extraction protocol. The preliminary results from the first phase of the study have been collected and showed that intra-laboratory variations were small, but large and significant interlaboratory variation was observed. The results were reported elsewhere. The preliminary results from the second part of the study consisting of the data currently collected are presented here. Again, intra-laboratory variations were small, but, also again, large and significant inter-laboratory variation was observed. However, in this part of the study, the range between the highest and lowest average results was narrower than in the first part of the study. Influence of the assay kit type was examined. The variation within assay kit type was small but significant differences in results were observed between assay kit types. The findings suggest that endotoxin concentration in samples can be ranked within laboratories, but not necessarily between laboratories. However, some of the variation between laboratories has been reduced by a common extraction protocol which suggests the possibility of further standardization that may lead to better comparability between laboratories.

Air Pollution, Indoor↗
PubMed1985

Comparative toxicity studies between bacterial lipopolysaccharide (endotoxin) and N-formyl methionyl peptide as factors in the pathogenesis of byssinosis.

Comparative in vivo and in vitro studies were made on bacterial lipopolysaccharide (LPS) and the chemotactic peptide NF-Met-Leu-Phe with a view toward studying their possible role in the pathophysiology of byssinosis. In contrast to LPS, chemotactic peptides did not cause Limulus amebocyte lysate gelation, nor did they induce the release of endogenous pyrogen. Inhalation of LPS caused a peripheral leukocytosis in rabbits 30 min after aerosol administration, whereas peptide inhalation caused a significant leukopenia in the same period. Cellular analysis of guinea pig bronchial lavages after LPS aerosol challenge revealed immediate decreases in all cell types, with subsequent, large increases of macrophages and granulocytes 4-24 h after aerosolization. Inhalation challenge with NF-Met-Leu-Phe induced no significant cellular changes. It was concluded that it is unlikely that these microbial products could be confused with each other when administered in pure form by the inhalation route.

Animals↗
PubMedDate not supplied

Effects of various treatments on the quantitative recovery of endotoxin from water-soluble metalworking fluids.

Three extraction methods were compared for their effectiveness in the quantitative removal of endotoxin from unused and used bulk water-soluble metalworking fluid (MWF) samples. Soluble, synthetic, and semisynthetic fluids were studied. The three modes of extraction consisted of (1) pyrogen-free water (PFW); (2) PFW and Tween 20 (polyoxyethylene sorbitan monolaurate); and (3) PFW, Tween 20, and sonication. Results suggest that vigorous recovery methods yield higher amounts of endotoxin from MWF samples than mild recovery methods in PFW alone. Additional studies are required to aid in the understanding of the factors that significantly affect endotoxin extraction yields from these fluids.

Aerosols↗
PubMed1986

The distribution of gram negative bacteria and endotoxin on raw cotton components.

The botanical composition of representative raw cottons from seven different growing regions was determined by manual removal and identification of all trash components greater than 50 micron in size. The number of gram negative bacteria (GNB) and the amount of endotoxin present in each of the separated raw cotton components were quantified. Low middling cotton contained significantly more bract-leaf trash than that found in higher quality cottons such as those in the middling grade division. Significantly more GNB and endotoxin were found in botanical trash components as well as lint of raw cotton derived from the southwest and southeast growing regions as compared to similar botanical components from far west cottons. For representative raw cottons from the 1980 USA crop we determined that 67% of the GNB and 89% of the endotoxin resided on white lint itself, from which all particulate larger than 50 micron in size had been removed manually.

Animals↗
PubMed1993

Inhibition and enhancement in the analysis of airborne endotoxin levels in various occupational environments.

Inhibition and enhancement effects on the Limulus Amebocyte Lysate (LAL) assay were studied in a series of endotoxin exposure samples from various occupational environments. Two types of analyses were performed, with various dilutions and with spiked samples. Results showed that inhibition and enhancement occurred in some samples, leading to respective underestimation and overestimation of the endotoxin concentration. When inhibition or enhancement is present, the result of this study shows that large differences in endotoxin concentration may be found, depending on the dilution at which the sample is analyzed. False conclusions about exposure levels and their potential health effects might be introduced. Results show that quality control procedures need to become standard practice when endotoxins are analyzed in occupational environments and development of a validated, generally accepted protocol should be encouraged.

Air Pollutants↗
PubMed1997

Environmental endotoxin measurement: interference and sources of variation in the Limulus assay of house dust.

Studies suggest that endotoxin levels in offices and homes may be associated with sick building syndrome or increased asthma severity. However, reported endotoxin levels in these studies were highly inconsistent, suggesting incompatible measurements from various laboratories. Therefore, an investigation of Limulus assay for endotoxin in house dust was undertaken. Interference with the assay was common and could produce endotoxin estimates varying by a factor of > 100, depending on the dilution used. Analysis of dose-response curves allowed detection of two types of interference: dilution-dependent and dilution-independent. Dilution-dependent interference persisted when samples were reassayed, but valid estimates could be obtained by appropriate dilution and data analysis. Valid estimates could not be obtained from assay showing dilution-independent interference. However, dilution-independent interference was frequently overcome by repeating the assay. Estimates based on a single sample dilution produced reasonable results on average when a dilution factor of 900 was used. However, the results were less precise from a single dilution (median coefficient of variation [CV] 40%) than from the complete dose-response curve (median CV 23%) and ranged from 11 to 240% of the estimates based on complete dose-response data. A comparison of extraction methods found that sonication in a phosphate-triethylamine buffer produced greater endotoxin activity than extraction in buffer with addition of saponin or sodium dodecyl sulfate. Endotoxin activity was stable for 8 to 10 weeks in dust samples stored at 4 and -20 degrees C but was not stable in extracts. The lot of Limulus amebocyte lysate reagent and method of detecting a response in the kinetic Limulus assay had significant effects on endotoxin estimates.

Analysis of Variance↗
PubMed1993

Assessment of ability of murine and human anti-lipid A monoclonal antibodies to bind and neutralize lipopolysaccharide.

The use of monoclonal antibodies (mAbs) directed to lipid A for the therapy of gram-negative sepsis is controversial. In an attempt to understand their biologic basis of action, we used a fluid-phase radioimmunoassay to measure binding between bacterial lipopolysaccharide (LPS) and two IgM mAbs directed to lipid A that are being evaluated for the treatment of gram-negative bacterial sepsis. Both antibodies bound 3H-LPS prepared from multiple strains of gram-negative bacteria when large excesses of antibody were used, although binding was modest and only slightly greater than control preparations. We also studied the ability of each anti-lipid A antibody to neutralize some of the biological effects of LPS in vitro. Despite large molar excesses, neither antibody neutralized LPS as assessed by the limulus lysate test, by a mitogenic assay for murine splenocytes, or by the production of cytokines interleukin (IL)-1, IL-6, or tumor necrosis factor from human monocytes in culture medium or in whole blood. Our experiments do not support the hypothesis that either of these anti-lipid A mAbs function by neutralizing the toxic effects of LPS.

Adult↗
PubMed1998

Limulus amebocyte lysate assay for detection of endotoxin in patients with sepsis syndrome. AMCC Sepsis Project Working Group.

Clinical predictions alone are insufficiently accurate to identify patients with specific types of bloodstream infection; laboratory assays might improve such predictions. Therefore, we performed a prospective cohort study of 356 episodes of sepsis syndrome and did Limulus amebocyte lysate (LAL) assays for endotoxin. The main outcome measures were bacteremia and infection due to gram-negative organisms; other types of infection were secondary outcomes. Assays were defined as positive if the result was > or = 0.4 enzyme-linked immunosorbent assay units per milliliter. There were positive assays in 119 (33%) of 356 episodes. Assay positivity correlated with the presence of fungal bloodstream infection (P < .003) but correlated negatively with the presence of gram-negative organisms in the bloodstream (P = .04). A trend toward higher rates of mortality in the LAL assay-positive episodes was no longer present after adjusting for severity. Thus, results of LAL assay did not correlate with the presence of bacteremia due to gram-negative organisms or with mortality after adjusting for severity but did correlate with the presence of fungal bloodstream infection.

Bacteremia↗
PubMed1995

Bacterial lipopolysaccharide copurifies with plasmid DNA: implications for animal models and human gene therapy.

During the course of gene therapy experiments in rodents, using intramuscular injections of plasmid DNA derived from Escherichia coli, we noted dose-related toxicity. This observation prompted a search for possible contaminants of DNA samples. We used the highly specific and sensitive limulus amoebocyte lysate assay (LAL), to monitor endotoxin bioactivity in DNA samples, and found plasmid DNA derived from standard E. coli bacterial strains, using traditional DNA isolation protocols, to be heavily contaminated with endotoxin, or lipopolysaccharide (LPA). Standard DNA isolation procedures resulted in the copurification of up to 500 micrograms/ml of LPS. LPS is a potent inducer of cytokines and other inflammatory mediators, and may complicate the use of naked DNA in gene therapy. The copurification of endotoxin with plasmid DNA also has important implications for in vitro transfection studies and microinjection of DNA into embryos. A simple and efficient protocol to reduce LPS contamination of plasmid DNA was developed. The conversion of intact bacteria to spheroplasts prior to the isolation of plasmid DNA, incubation with lysozyme, treatment with the detergent n-octyl-beta-D-thioglucopyranoside (OSPG) and polymyxin-B (PMB) chromatography, allowed the isolation of plasmid DNA containing less than 50 ng/ml LPS. This represents a 10,000-fold reduction in LPS contamination, compared to conventional methods of plasmid DNA purification, avoids potentially toxic reagents such as ethidium bromide, and produces a higher yield of plasmid DNA.

Animals↗
PubMed1998

Validation of a quality assurance program for autologous cultured chondrocyte implantation.

The use of living human cells to facilitate repair of defects in structural tissues is a rapidly emerging treatment option. A quality assurance program based on U.S. Food and Drug Administration good manufacturing practice regulations and other guidance was established and implemented in a program to use autologous cultured chondrocytes for repair of articular defects of the knee. The development of ex vivo cell therapies presents novel issues of quality assurance. The implementation and evaluation of this quality program was based on the implantation of 303 patients with autologous cultured chondrocytes, in which we analyzed a number of quantifiable parameters and which meets the unique challenges of autologous cell therapy within a rigorous regulatory framework. Application of well-accepted principles of quality assurance and quality control coupled with a thorough understanding of the cell culturing processes will result in a safe and efficacious cell therapy product.

Cartilage, Articular↗
PubMed1992

Antibiotic-induced release of endotoxin: a reappraisal.

A three- to 20-fold increase in the total concentration of endotoxin occurs as a consequence of antibiotic action on gram-negative bacteria both in vitro and in vivo. There is considerable overlap between the effect of beta-lactam antibiotics and non-beta-lactam antibiotics. Moreover, there is an unexplained delay between the lethal activity of antibiotics and the release of endotoxin. Hence, the mechanism whereby antibiotic action leads to the release of endotoxin is unclear, and mechanisms other than bacterial lysis warrant consideration. The evidence that the release of endotoxin has clinical importance is conflicting, and the issue is unresolved. However, nonlytic release may have implications for the therapeutic efficacy of antiendotoxin immunotherapy. Although frequently cited in the context of the antibiotic-induced release of endotoxin, a number of important differences pertain to conditions, such as the Jarisch-Herxheimer reaction and the tumor lysis syndrome, for which there is clear evidence of an initial deterioration with effective therapy.

Animals↗
PubMedDate not supplied

Mitogenic activities of lipid A and liposome-associated lipid A: effects of epitope density.

Among eight purified lipid A fractions, there was heterogeneity of mitogenic activity, but the relative activity could not be predicted by limulus amebocyte lysate assay. Analysis of mitogenic activity after the incorporation of lipid A into liposomes demonstrated that epitope density of lipid A was a crucial factor that strongly modulated expression of lipid A. The results suggest that lipid A hydrophobic groups are responsible for mitogenic activity and that the surface accessibility of lipid A hydrophobic groups in membranes may be influenced by epitope density. The data are compared with previous results derived from parallel studies on limulus amebocytes. Both approaches lead to the conclusion that the activities of purified lipid A fractions are heterogeneous and that epitope density is critically important for the expression of biologic activity of membrane-associated lipid A.

Animals↗
PubMed1975

Effect of heparin in experimental gram-negative septicemia.

Young rabbits were given live encapsulated or heat-hilled Pasteurella multocida by the intraperitoneal route. Those animals given live organisms regularly developed clinical illness, positive blood cultures, positive limulus lysate tests on plasma, leukopenia, thrombocytopenia, and disseminated intravascular coagulation and died. Death occurred in 68% of these rabbits by 12 hr after infection and in 100% by 24 hr. Septicemic animals given heparin as an anticoagulant similarly developed clinical illness, leukopenia, and thrombocytopenia, but no laboratory evidence of disseminated intravascular coagulation. The mortality rate in these animals was 90% 12 hr after infection and 100% 24 hr after infection. None of the animals given heat-killed organisms developed leukopenia, thormbocytopenia, or disseminated intravascular coagulation, and none died. The data show that the survivial rate in septicemic rabbits was not improved with heparin, although fibrinogen consumption (disseminated intravascular coagulation) was abolished.

Animals↗