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PubMed1990

Comparison of cultured human mononuclear cells, Limulus amebocyte lysate and rabbits in the detection of pyrogens.

Isolated human mononuclear cells exposed to either lipopolysaccharide or Staphylococcus aureus secreted interleukin-1 like material. The secretion was concentration dependent. The minimal detectable level in the test solution of lipopolysaccharide and Staphylococcus aureus was 200 pg/ml and 10(5) cells/ml respectively. The sensitivity and specificity of the Limulus Amebocyte Lysate test, the rabbit pyrogen test and the monocyte test are compared. The monocyte test is proposed as an alternative in-vitro test to the rabbit pyrogen test.

Animals↗
PubMed1986

Chemical composition and biological properties of a lipopolysaccharide from Bacteroides intermedius.

Endotoxin (lipopolysaccharide, LPS) from an oral strain of Bacteroides intermedius was isolated by phenol extraction and purified by ultracentrifugation and gel filtration. The preparation was essentially free from contaminating nucleic acid and protein. The LPS contained rhamnose, fucose, mannose, glucose, galactose, glucosamine, and an unidentified sugar (approximate molar ratios 9:1:6:3:1:7:2). Neither heptose nor 2-keto-3-deoxyoctonate was detected. The major fatty acids were 3-hydroxy-15-methylhexadecanoic acid and 3-hydroxyhexadecanoic acid. The LPS was homogeneous with regard to molecular size, and its polysaccharide chain appeared short compared to the E. coli 055 LPS which was used as reference. A molecular weight of approximately 7,800 was estimated from gas chromatography data and by gel filtration in the presence of sodium deoxycholate. The B. intermedius LPS demonstrated low potency in the Limulus amoebocyte lysate assay, and in the chick embryo and mouse lethality tests and gave negative response in the local Shwartzman reaction.

Bacterial Toxins↗
PubMed1994

Effect of human recombinant cytokines on the induction of macrophage procoagulant activity.

A panel of human recombinant cytokines was tested for induction of procoagulant activity (PCA) in human monocyte-derived macrophages. Nonadherent culture conditions were used, and PCA was determined with whole cells rather than cell lysates. It was assured by Limulus amebocyte lysate assay that tested cytokines displayed low levels of endotoxin activity within the range of biologic activity. Additional evidence to rule out an endotoxin effect was provided by heat-inactivation experiments. Interferon-gamma (IFN-gamma), interleukin-1 beta (IL-1 beta), and tumor necrosis factor-alpha (TNF-alpha) were strong macrophage PCA inducers. The low level of PCA induced by IL-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), M-CSF, IL-4, IL-6, IL-10, and IFN-alpha could not be distinguished from that induced by traces of endotoxin contaminating the preparations. Transforming growth factor-beta decreased constitutively expressed PCA within 24 hours of exposure. PCA induced by IFN-gamma, IL-1 beta, and TNF-alpha depended largely on tissue factor expression, as evidenced by experiments with factor X-deficient plasma and antitissue factor antibodies. In macrophages subcultured in adherence, IL-1 beta was a strong PCA inducer, whereas IFN-gamma and TNF-alpha promoted little PCA increase. This observation and different kinetics of PCA induction suggested that mechanisms of PCA induction are distinct for the three cytokines. Thus, we showed that well-characterized cytokines critically involved in the promotion of cell-mediated antimicrobial defense/delayed-type hypersensitivity and considered for clinical application promote local fibrin deposition by a direct effect on macrophages.

Blood Coagulation Factors↗
PubMed1985

Chromogenic Limulus amoebocyte lysate assay for rapid detection of gram-negative bacteriuria.

A chromogenic Limulus amoebocyte lysate assay was evaluated as a rapid screening test for the detection of clinically significant gram-negative bacteriuria. The development of a distinctive yellow color after the addition of chromogenic substrate to the Limulus amoebocyte lysate-urine reaction mixture was used to measure greater than or equal to 10(5) gram-negative bacteria per ml. A total of 324 urine specimens were assayed, with 68 gram-negative urinary tract infections identified as defined by quantitative urine colony counts of greater than or equal to 10(5) bacteria per ml. Of these, 68 and 67 of 68 were detected by the chromogenic Limulus amoebocyte lysate assay at urine dilutions of 1:10 and 1:20, respectively. Nine false-positive chromogenic Limulus amoebocyte lysate assay results were observed at both urine dilutions and in the same specimens. At a urine dilution of 1:10, sensitivity and specificity were 100 and 96.6%, respectively, with predictive values of 100% for a negative test and 88.3% for a positive test. At a urine dilution of 1:20, sensitivity and specificity were 98.6 and 96.6%, respectively; predictive values were 99.6% for a negative test and 88.3% for a positive test. These data suggest that chromogenic Limulus amoebocyte lysate assay of urine has potential usefulness as a rapid, reliable, and easily performed and interpreted screening test for the diagnosis of clinically significant gram-negative bacteriuria.

Bacteriuria↗
PubMed1997

Human monocytoid cell lines as indicators of endotoxin: comparison with rabbit pyrogen and Limulus amoebocyte lysate assay.

The aim of this study was to develop an in vitro test system for pyrogenic substances. Three clones derived from human monocytoid cell lines, which were selected by their high sensitivity to lipopolysaccharide (LPS), were assessed for tumor necrosis factor (TNF) production. Their response to pyrogen-containing samples was compared with that in a Limulus amoebocyte lysate assay and the rabbit pyrogen test. We show here that the induction of TNF in these clones is a valid in vitro alternative to determine endotoxin in commercial preparations requiring pyrogenicity testing. Cell clones derived from Mono Mac 6 (MM6 2H8 and MM6 4B5) responded to sub-ng/ml concentrations of complete rough-strain and smooth-strain LPS, to ng/ml concentrations of diphosphoryl-lipid A, and to microgram/ml concentrations of monophosphoryl-lipid A and to detoxified LPS. Cells reacted to > or = 1 microgram/ml lipoteichoic acid by TNF production, and were relatively insensitive to toxic shock syndrome toxin-1 (TSST-1) and to muramyl dipeptide adjuvant peptide. The reaction pattern of a clone derived from THP-1 (THP-1 1G3) was in general, similar to that of the MM6 clones, except that THP-1 1G3 failed to react to diphosphoryl-lipid A. When tested on commercial samples destined for parenteral use, there was a close correlation between a sensitive Limulus amoebocyte lysate (LAL) test and the cell culture test on the one hand, and between the pyrogen test and the cell culture test on the other hand. The data suggest that this cell-based test is able to recognize pyrogens derived from gram-negative organisms in test samples with appropriate sensitivity and specificity. This test appears to be able to eliminate some of the false-positive data obtained in the LAL test.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1987

Recovery of endotoxin from human plasma by acid oxidative treatments as monitored by an automated microtiter plate-chromogenic substrate Limulus amebocyte lysate (LAL) assay method.

Methods to detect endotoxin in human plasma for LAL testing have been developed by use of a perchloric acid (PCA) or a trifluoroacetic acid (TFA) treatment. These acid oxidative treatments eliminate interferences present in plasma. The mode of action of the acids for the elimination of the interferences is indicated to be by their oxidative actions. The treated plasma is assayed by use of an automated microtiter plate-chromogenic substrate LAL assay method employing a modified Cetus Pro/Pette EXPRESS system. The automated system assays 96 samples in approximately 22 minutes. Assay sensitivity of the automated Pro/Pette system is approximately 0.01 endotoxin units (EU) per ml with a linear range from 0.03 to 0.14 EU/ml. The relative standard deviation (RSD) of the assay system is approximately 6%. The improved precision of the LAL assay by the automated system has been attributed to its fast speed (2 minutes) in dispensing the unstable LAL reagent to all 96 samples in one assay block. The total operation, including the acid oxidative treatments and the 96 sample LAL assay, takes approximately 45 minutes. The recovery of spiked endotoxin from plasma ranged from 75 to approximately 100% by adding 0.4 ml of either 0.1-0.2 N PCA or 0.2-0.35 N TFA to 0.2 ml plasma and reacting for 15 minutes at 37 degrees C. The standard deviation of the assay is approximately 0.1 EU/ml plasma. The method has been proven to be rugged, simple, rapid, and cost effective thus would be suitable for clinical application.

Autoanalysis↗
PubMed1984

Biological activities of synthetic lipid A analogs: pyrogenicity, lethal toxicity, anticomplement activity, and induction of gelation of Limulus amoebocyte lysate.

Chemically synthesized lipid A analogs were investigated for several endotoxic activities, including pyrogenicity, lethal toxicity, anticomplement activity, and the capacity to gelate Limulus amoebocyte lysate in comparison to natural lipid A. The synthetic preparations contained D-glucosamine or D-glucosamine-beta-1,6-D-glucosamine disaccharide substituted by ester- and amide-bound hydroxylated or non-hydroxylated fatty acids and by phosphate groups in different combinations. Some preparations which were insoluble in water were succinylated and thus rendered more soluble. Strong biphasic pyrogenic responses with a maximal increase in body temperature of 1 to 2 degrees C were obtained with 50 micrograms/kg doses of 3 disaccharide preparations of 15 tested. With two preparations (50 micrograms/kg) moderate pyrogenicity with monophasic fever curves and a maximal temperature increase of about 0.6 degrees C was obtained. Lethal toxicity tests were carried out in galactosamine-sensitized mice. Of 15 synthetic preparations, 4 exhibited lethal toxicity under these conditions. The effective doses of the lipid A analogs in both in vivo tests were, however, several hundred times higher than those of bacterial lipid A. For the activities in vivo, hydroxyacyl residues seemed to be important. Anticomplement activity was demonstrable in seven preparations, one of which expressed an activity comparable to that of lipid A. Preparations containing non-hydroxylated fatty acids seemed to be most active in this test. None of the synthetic preparations was found to exhibit gelation activity for Limulus amoebocyte lysate when tested in doses up to 0.4 micrograms, whereas bacterial free lipid A was active in doses of about 2 pg. None of the monosaccharide derivatives exhibited any of these activities.

Animals↗
PubMed1997

Comparison between Wako-WB003 and Fungitec G tests for detection of (1-->3)-beta-D-glucan in systemic mycosis.

The limulus factor G reacts with (1-->3)-beta-D-glucan, a major structural component of fungal cell walls. The Fungitec G test is a colorimetric assay that measures the concentration of (1-->3)-beta-D-glucan and is used as a serodiagnostic test for deep mycosis. Wako-WB003 is another assay for (1-->3)-beta-D-glucan that determines the change in turbidity of the gelatin reaction of limulus factor G with (1-->3)-beta-D-glucan. In five rabbits inoculated intravenously with 1 x 10(7) CFU of Candida albicans, the concentration of (1-->3)-beta-D-glucan measured by the fungitec G test increased gradually reaching a peak of 660.9 +/- 427.9 pg/ml (mean +/- SD) 4 days after inoculation, but to 42.225 +/- 41.275 ng/ml on day 6 in the Wako-WB003 test. In one rabbit challenged intravenously with 5 x 10(6) CFU of C. albicans, (1-->3)-beta-D-glucan increased to 101.5 pg/ml on day 4 on the fungitec G test, whereas the level remained below the detection limit of the Wako-WB003 test throughout the course of the disease. We also detected high concentrations of (1-->3)-beta-D-glucan in 11 patients with candidemia, 4 with suspected candidemia, 1 with invasive pulmonary aspergillosis, and 12 patients with aspergilloma. The concentration of (1-->3)-beta-D-glucan measured by the Fungitec G test was > 150, > 1006.8; 312.1, and 55.6 +/- 37.4 pg/ml (range, 20.1-138.0 pg/ml), and by the Wako-WB003 test > 153.000, > 17.70, 153.000 and 2.645 +/- 7.248 ng/ml (range, < 25.20 ng/ml) in these patients, respectively. In contrast, the concentration of (1-->3)-beta-D-glucan in 9 patients with pulmonary cryptococcosis and 6 with superficial candida colonization ranged from < 13.2 and < 15.3 pg/ml in the Fungitec G test and < 0.53 and < 0.12 ng/ml in Wako-WB003 test. There was a weak relationship between the concentration of (1-->3)-beta-D-glucan measured by the Fungitec G test and Wako-WB003 test (r = 0.521). Our results indicate that the sensitivity of the Wako-WB003 test is lower than that of the Fungitec G test.

Animals↗
PubMed1985

U.S. Government quality control program for Limulus amebocyte lysate and endotoxin.

The U.S. Government has licensed seven Limulus Amebocyte Lysate manufacturers. Each manufacturer must determine the sensitivity of each lot by using the U.S. Standard Endotoxin, EC-5 which is identical to the U.S. Pharmacopeia (USP) Endotoxin Standard (Lot F). A licensed firm must be inspected annually and submit a protocol along with potency testing results on each lot of lysate before it can be released. The FDA tests each lot for at least potency before releasing it to be marketed. The release criteria for both the gelation and chromogenic lysate tests are discussed.

Chromogenic Compounds↗
PubMed1976

Detection of bacterial endotoxin in antitumor agents.

The Limulus amebocyte lysate assay was used to detect the presence of bacterial endotoxin in a variety of chemotherapeutic agents currently in use. Results were quantitated using a standard Escherichia coli endotoxin curve. Relative concentrations of endotoxin ranging from 0.1 to 63 ng/ml were detected in individual lots of L-asparaginase, 5-azacytidine, bleomycin, DTIC, actinomycin D, adriamycin, and vinblastine diluted to normal concentrations for injection. When multiple lots of a given drug were tested, at least one lot produced a negative assay, indicating less than 0.1 ng/ml of endotoxin in 50% of these drugs. All lots of cytosine arabinoside, cyclophosphamide, daunorubicin, vincristine, and streptozotocin tested had less than 0.1 ng/ml of endotoxin. Inactivation kinetics of the Limulus-positive drug material in dilute alkali solutions were identical to those of E. coli endotoxin. The Limulus lysate test is a rapid and specific method for the detection of small amounts of bacterial endotoxin contaminating parenteral preparations of antitumor agents.

Animals↗
PubMed1979

Biological activity of the slime and endotoxin of the periodontopathic organism Eikenella corrodens.

A partially purified water-soluble slime extract was obtained from two strains of the oral pathogen Eikenella corrodens, designated CS10 A and CS10 B. Endotoxin was also isolated from this organism by the phenol-water extraction procedure. Assays including the local Shwartzman skin reactivity, chicken embryo lethality, Limulus lysate clotting, spleen cell mitogenicity, and immune adjuvancy were used to test the biological properties of these two bacterial extracts. In each of these assay systems the endotoxins of the Eikenella corrodens strains demonstrated the classical endotoxic responses. In contrast, the effect of slime extract in the corresponding assays was either extremely low or absent with the exception of a very strong immunosuppressive effect. Mice treated with slime extract showed a severely reduced immune response to sheep erythrocytes as measured by the hemolytic plaque-forming cell assay.

Animals↗
PubMed1985

Ultrafiltration as a means to eliminate inhibition of the Limulus Amoebocyte Lysate (LAL) assay.

Inhibition of the Limulus Amoebocyte Lysate (LAL) assay for the detection of endotoxin is often a problem when testing parenteral solutions with actual concentrations of chemical substances. Seven samples of sterile, pyrogen-free solutions widely used in Norwegian hospitals, with inhibiting effect on the LAL test, were spiked with endotoxin, and ultrafiltered with different volumes of diluent. For six of the seven samples the inhibition was reduced or eliminated.

Limulus Test↗
PubMed1989

A method for removing interleukin-1- and tumor necrosis factor-inducing substances from bacterial cultures by ultrafiltration with polysulfone.

The ability of human mononuclear cells (MNC) to produce cytokines is a highly sensitive and biologically relevant test system for the presence of microbial products. The safety of parenteral fluids is presently determined by gelation of the limulus amebocyte lysate (LAL) to endotoxin. In the present study, crude bacterial culture supernatants from Escherichia coli were subjected to ultrafiltration using polysulfone and the ultrafiltrates were tested for their ability to stimulate human MNC. Total interleukin-1 (IL-1) and tumor necrosis factor (TNF) produced by MNC were measured by radioimmunoassay. Endotoxin-like substances in E. coli cultures are rejected by a factor of at least 100,000. Rejection takes place by molecular size exclusion and by absorption. The sensitivity of the LAL and MNC cytokine production were comparable. These studies demonstrate a wide margin of safety for the production of parenteral fluids using ultrafiltration for endotoxin-containing materials.

Bacterial Toxins↗
PubMed1982

Gelatin of Limulus amoebocyte lysate by simple polysaccharides.

The Limulus lysate gelatin activity of several simple polysaccharides including yeast mannans and bacterial dextrans was investigated. The mannans from Saccharomyces cerevisiae wild and mutant strains possessing dense branches showed positive gelatin activity at concentrations of 1 microgram/ml or more regardless of differences in their chemical structure. However, two synthetic mannans possessing linear structures with alpha 1 leads to 2 and alpha 1 leads to 6 linkages also gave positive reactions at concentrations of 10 microgram/ml or more and 500 microgram/ml or more, respectively. The dextran from Leuconostoc mesenteroides IAM 1046 consisting of a dense branching moiety displayed reactivity at concentrations of 100 microgram/ml or more, while the dextrans devoid of such branches were negative in this reaction. The optimal concentration for Limulus lysate gelatin could not be determined for any of the polysaccharides and lipopolysaccharides (LPS) tested in this study. The gelation activity of the polysaccharides was stable to treatment with 100 mM NaOH at 30 C for 72 hr. The minimum concentration for the gelation activity of LPS treated with 100 mM NaOH under the same conditions was reduced from 10(-6) approximately(-9) microgram/ml to 1-10 microgram/ml. The above findings demonstrate that the major part of Limulus lysate gelation activity of LPS depends on the alkali-degradable lipid A moiety, and that such simple polysaccharides are also able to participate in this activity even though the extent of participation is very low.

Alkalies↗
PubMed1997

A novel inhibitor of bacterial endotoxin derived from cinnamon bark.

A substance that inhibits the activity of bacterial endotoxin (LPS) was found in cinnamon bark. The inhibitor, extracted from dry cinnamon bark with 67% ethanol/water, was purified by using Limulus gelation activity as an indicator of endotoxin activity. The inhibitor suppressed the activity of the LPS when it was mixed with the inhibitor prior to the assay. The reduction of the LPS activity depended on the concentration of both the inhibitor and LPS when mixed, and also on the incubation time. The inhibitor suppressed the activity of all LPS and lipid A preparations tested regardless of the origin of the bacteria. The inhibitor alone did not affect the Limulus system. These results indicate that the inhibition was caused by direct interaction of the inhibitor with the LPS molecule. Furthermore the inhibitor abrogated the pyrogenicity of the LPS. Although it is uncertain whether the inhibitor actually plays a role in the defense mechanism in cinnamon bark, this is the first report that an inhibitor of bacterial endotoxin exists in a plant.

Animals↗
PubMed1986

Endotoxin-inactivating activity in normal and pathological human blood samples.

The endotoxin-specific chromogenic test revealed that plasma endotoxin-inactivating activity was markedly diminished by endotoxemia, but not by fungemia or by dialysis with cellulose membranes, suggesting that fungal polysaccharides and other nonendotoxic, Limulus-reactive materials do not consume endotoxin-inactivating factors in the blood. There was a close negative correlation between plasma endotoxin concentration and endotoxin-inactivating activity. The specificity of the test was improved by fractionating amebocyte lysate and using only the factors that constitute the endotoxin-sensitive coagulation pathway of the horseshoe crab. This test was able to differentiate endotoxemia from fungemia and from contamination with other nonendotoxic, Limulus-reactive materials.

Endotoxins↗
PubMed1979

Limulus amebocyte lysate - a means to monitor inactivation of lipopolysaccharide.

A properly selected Limulus Amebocyte Lysate (LAL) has been demonstrated to have a good correlation with the in vivo United States Pharmacopeial pyrogen test when testing lipopolysaccharide (LPS) inactivated with ethylene oxide, 60 Co irradiation, and dry-heat treatments. Thus, LAL may be used to determine degradation or depyrogenation kinetics of LPS. A criteria for acceptance of an LAL lot for correlation with the USP pyrogen test is described.

Animals↗
PubMed1984

Influence of fine structure of lipid A on Limulus amebocyte lysate clotting and toxic activities.

We examined the relationship between the fine structure of lipid A and the toxicity of endotoxin or lipopolysaccharides as measured by the Limulus amebocyte lysate (LAL), rabbit pyrogenicity, chicken embryo lethal dose, and dermal Shwartzman reaction tests. Lipid A and lipid A-like compounds obtained from deep-rough mutants of Salmonella spp. and Escherichia coli had a wide range of structural variations. These compounds included native lipopolysaccharides, diphosphoryl and monophosphoryl lipid A's, and lipid X (a monosaccharide). The LAL test was positive for all lipids tested with lysates from Travenol Laboratories and from Associates of Cape Cod (2.9 X 10(3) to 2.6 X 10(7) endotoxin units per mg), except for O-deacylated and dephosphorylated lipid X, which were negative. The Mallinckrodt lysate gave negative tests for lipid X. In the rabbit pyrogenicity and chicken embryo lethal dose tests, only native lipopolysaccharide and diphosphoryl lipid A's were judged toxic. The Shwartzman reaction was positive for a specific purified diphosphoryl lipid A (thin-layer chromatography-3 fraction) but negative for the purified monophosphoryl lipid A (also a thin-layer chromatography-3 fraction). These results show that the LAL test is not a valid measure of all parameters of toxicity of a lipid A or lipid A-like compound and can yield false-positive results. However, these findings are not in conflict with the widespread use of the LAL assay for pyrogens in the pharmaceutical industry since a good correlation exists between LAL results and pyrogenicity when undegraded endotoxin is evaluated in parallel assays.

Animals↗