Limulus Amoebocyte Lysate Gelation Research Articles

One Week of Nitrofurantoin Before Percutaneous Nephrolithotomy Significantly Reduces Upper Tract Infection and Urosepsis: A Prospective Controlled Study

To evaluate the role of nitrofurantoin (NFT) prophylaxis in a prospective randomized control study. Urosepsis is an important complication after percutaneous nephrolithotomy (PNL). Risk increases by around 4 times with larger stones and hydronephrosis (HDN). Patients with stones ≥2.5 cm and/or HDN and sterile urine undergoing PNL were randomized into 2 groups. Standard perioperative antibiotic prophylaxis was the same in both groups. One group received sustained-released NFT 100 mg b.i.d. for 7 days preoperatively, and the other did not. Preoperative urine, intraoperative renal pelvic urine, and stone cultures were obtained. Postoperative occurrence of SIRS was considered urosepsis after excluding other causes. Serum samples were collected immediately after PNL and stored at -20°C. Serum endotoxin was estimated using Limulus Amoebocyte Lysate gelation technique (Sigma Aldrich, Saint Louis, Missouri). The operating surgeons and the microbiologist were blinded to the group distribution. Of 101 patients, 48 received nitrofurantoin prophylaxis. Both groups were comparable for age, gender, stone burden, degree of HDN, duration of operation, and intraoperative blood loss. There was significantly low positive pelvic urine culture (0% vs 9.8%, RR 4.95, P = .001), positive stone culture (8.3% vs 30.2%, RR 3.64, P = .001), endotoxemia (17.5% vs 41.9%, OR 0.22, P = .016), and systemic inflammatory response system (19% vs 49%, OR 0.31, P = .01) in patients receiving NFT prophylaxis. Prophylaxis with NFT for a week before PNL is beneficial in the prevention of urosepsis and endotoxemia in patients with larger stones and HDN. NFT covers most of the urinary isolates and is preferred in areas of fluoroquinolone resistance.

Structure of Compositionally Simple Lipopolysaccharide from Marine Synechococcus

Lipopolysaccharide (LPS) is the first defense against changing environmental factors for many bacteria. Here, we report the first structure of the LPS from cyanobacteria based on two strains of marine Synechococcus, WH8102 and CC9311. While enteric LPS contains some of the most complex carbohydrate residues in nature, the full-length versions of these cyanobacterial LPSs have neither heptose nor 3-deoxy-D-manno-octulosonic acid (Kdo) but instead 4-linked glucose as their main saccharide component, with low levels of glucosamine and galacturonic acid also present. Matrix-assisted laser desorption ionization mass spectrometry of the intact minimal core LPS reveals triacylated and tetraacylated structures having a heterogeneous mix of both hydroxylated and nonhydroxylated fatty acids connected to the diglucosamine backbone and a predominantly glucose outer core-like region for both strains. WH8102 incorporated rhamnose in this region as well, contributing to differences in sugar composition and possibly nutritional differences between the strains. In contrast to enteric lipid A, which can be liberated from LPS by mild acid hydrolysis, lipid A from these organisms could be produced by only two novel procedures: triethylamine-assisted periodate oxidation and acetolysis. The lipid A contains odd-chain hydroxylated fatty acids, lacks phosphate, and contains a single galacturonic acid. The LPS lacks any limulus amoebocyte lysate gelation activity. The highly simplified nature of LPSs from these organisms leads us to believe that they may represent either a primordial structure or an adaptation to the relatively higher salt and potentially growth-limiting phosphate levels in marine environments.

Detection of Pyrogens in Intravenous IgG Preparations

Five different intravenous IgG (i.v. IgG) preparations were assessed for their capacity to modify the pyrogenic response to bacterial lipopolysaccharide (LPS) of rabbits under the conditions of a pharmacopoeal test. Four of the five preparations were found to mitigate the reaction rendering the result “non-pyrogenic” with an LPS dose proved pyrogenic when administered in saline or in albumin. Bacterial LPS was found readily detectable by a simple Limulus amoebocyte lysate (LAL) gelation test. Four of six brands of i.v. IgG were found reactive in the test under conditions adjusted to detect the FDA limit. The reaction obtained upon addition of standard LPS to the negative preparations supported the validity of the assay. The LAL reactivity of two of the reactive preparations was inhibited by laminarin, a compound known to inhibit Limulus lysate gelation by β-D-glucan, but not by Polymyxin B. Specific detection of bacterial endotoxins in i.v. IgG solutions requires inhibition of the β-D-glucan pathway of the Limulus lysate coagulation. Using an appropriate inhibitor, the LAL gelation test is suitable to detect a potential endotoxin contamination in i.v. IgG which might have not been unravelled by the in vivo test for pyrogens. Copyright 2002 Published by Elsevier Science Ltd on behalf of The International Association for Biologicals

Dissociation of endotoxic activities in a chemically synthesized lipid A precursor after acetylation.

In a previous study, a chemically synthesized disaccharide precursor of lipid A (406; identical to lipid IVA) was shown to have dramatically reduced lethality, B-cell mitogenicity, and tumor necrosis factor induction in macrophages when its hydroxyl groups were replaced with either succinyl or acetyl residues (K. Tanamoto, FEBS Lett. 351:325-329, 1994). Succinylated 406 was found to lose Limulus amoebocyte lysate gelation activity completely as a result of the modification (about 10(5)-fold), too, as expected. However, acetyl 406, surprisingly, exhibited activity comparable to that of the original 406. Both succinylated and acetylated 406 lost pyrogenicity completely. These results indicate that one of the typical endotoxic activities was dissociated from the others and that the ability to induce Limulus amoebocyte lysate gelation is not always representative of endotoxin activity.

Anti-lipopolysaccharide activity of histatins, peptides from human saliva.

Histatins are histidine-rich polypeptides secreted in human saliva. They were found to inhibit lipopolysaccharide (LPS)-mediated gelation of Limulus amoebocyte lysate, and to reverse the anti-complement action of LPS or lipid A. Histatins also gave precipitate bands in agarose gels with various LPS. The results indicate that histatins neutralized the activity of LPS by binding to the lipid A moiety of LPS.

Comparative immunochemistry of lipopolysaccharides from Branhamella catarrhalis strains

Lipopolysaccharides (LPS) were extracted and purified from the type strain and from a clinical isolate of Branhamella catarrhalis. Chemical analysis revealed the presence of glucose, galactose, and glucosamine in different molar proportions in the LPS from these two isolates, whereas there was no difference between the two isolates in the ratios of ketodeoxyoctonate, phosphate, and the fatty acids C12, 3-OH-C12, and 3-OH-C11 present. Heptose or 3-OH-C14 was not detectable in either preparation. LPS from both strains appeared semirough according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, presenting a core polysaccharide plus one repeating unit. Immunoblotting, passive hemolysis, and hemolysis inhibition assays using anti-LPS antibodies from immunized rabbits demonstrated cross-reactivity between the LPS preparations; however, antigenic dissimilarities were also found, suggesting that more than one serotype may exist. The lipid A isolated from the two LPS was serologically identical and exhibited cross-reactivity with lipid A of members of the family Enterobacteriaceae. The B. catarrhalis LPS were biologically active, causing lethality in D-galactosamine-sensitized C57/BL6 mice and inducing Limulus amoebocyte lysate gelation.

Gelation ofLimulus amoebocyteLysate by Glucans

Gelation of<i>Limulus amoebocyte</i>Lysate by Glucans

Measurements of lipopolysaccharide (endotoxin) in meningococcal protein and polysaccharide preparations for vaccine usage

Lipopolysaccharide (LPS, i.e. endotoxin) present in meningococcal outer-membrane protein and polysaccharide preparations made for vaccine use was quantitated by a silver-stain method following SDS-PAGE. The reactivities of LPS in the preparations were also measured by rabbit pyrogenicity and Limulus amoebocyte lysate (LAL) assay. Although rabbit pyrogenicity and LAL assay are more sensitive than the silver stain method, the latter provided an actual amount of LPS present in the protein or in the polysaccharide. For a meningococcal protein preparation, rabbit pyrogenicity showed about one-tenth, and even less by LAL assay, of the actual amount of LPS. This is because protein-bound LPS in meningococcal protein preparations is about 10-fold less active in causing fever in rabbits, and 20- to 40-fold less active in the gelation of LAL than the same amount of a purified free LPS which is generally used as a reference in quantitating LPS in these two assays. As for the small amount of LPS present in a meningococcal polysaccharide preparation, similar LPS content was obtained when measured by the three methods suggesting that the LPS is not bound to the polysaccharide in contrast to that in the proteins mentioned above. The purified meningococcal LPS was pyrogenic in rabbits at 1 ng/kg.

The International Standard for Endotoxin: evaluation in an international collaborative study

An ampouled preparation of bacterial endotoxin, coded 84/650, was evaluated in 35 laboratories in 12 countries for its suitability to serve as the International Standard for Endotoxin. The ampouled preparation was calibrated in terms of the USA National Standard, EC5, in Limulus Amoebocyte Lysate gelation, turbidimetric and chromogenic tests and in rabbit pyrogen tests. On the basis of the results reported here, with the agreement of the participants in the study, and with the authorization of the Expert Committee on Biological Standardization of the World Health Organization, the preparation coded 84/650 was established in 1986 as the International Standard for Endotoxin for Limulus Gelation Tests with an assigned unitage of 14 000 IU of endotoxin per ampoule.

A procedure for the efficient incorporation of wild-type lipopolysaccharide into liposomes for use in immunological studies

Previous studies on the mechanism of action of lipopolysaccharides (LPS) on macrophages have used wild-type lipopolysaccharide (wt-LPS) containing liposomes. In these studies the endotoxin was incorporated into liposomes by suspending the wt-LPS in the buffer used to rehydrate thelipid. Using this approach (buffer method), we observed that less than 10% of Salmonella minnesota smooth LPS is incorporated into multimellar vesicles (MLV). If the non-incorporated material is not effectively separated from the liposomal form, erroneous conclusions on the mechanism of action of LPS can be drawn. Prolonged sonication of the wt-LPS-MLV suspension resulted in almost complete incorporation of the LPS into the resulting small unilamellar vesicles (SUV). In order to prepare MLV, we briefly soniated the buffer preparation, dehydrated the resulting smaller vesicles and then rehydrated the mixture (dry method). This procedure resulted in almost complete incorporation of the wt-LPS into MLV. The ability of wt-LPS in MLV prepared by the dry method to activate macrophages or trigger gelation of Limulus amoebocyte lysate was reduced by 100–1000-fold compared to the non-incorporated wt-LPS. This indicates that at least 99% of the wt-LPS is incorporated in MLV made by the dry method.

Binding and neutralization of bacterial lipopolysaccharide by colistin nonapeptide

Polymyxin nonapeptides, proteolytic derivatives of polymyxin antibiotics, are less toxic than their parent compounds but retain some of their antibacterial activities. To confirm and expand observations that polymyxin nonapeptides have anti-endotoxin activity, we studied the ability of colistin nonapeptide to bind to bacterial lipopolysaccharide (LPS) and to inhibit the effects of LPS on Limulus amoebocyte lysate and lymphocyte mitogenicity. Colistin nonapeptide was purified by high-pressure liquid chromatography and was demonstrated to bind to LPS by equilibrium dialysis. The ability of colistin nonapeptide to render E. coli ATCC 25922 cells sensitive to erythromycin was abrogated by 50% after incubation with E. coli O18 LPS in a ratio by weight of LPS to colistin nonapeptide of 3.9:1. The presence of 4 micrograms of colistin nonapeptide or colistin per ml increased by 130- and 800-fold, respectively, the concentration of E. coli O113 LPS required to produce 50% gelation of Limulus amoebocyte lysate as measured by a spectrophotometric assay. Neutralization of LPS by colistin nonapeptide was time and concentration dependent. In contrast to the neutralization seen with LPS derived from a colistin-sensitive organism, colistin nonapeptide neutralized very little LPS extracted from a strain of Serratia marcescens that was resistant to colistin. Colistin nonapeptide also inhibited LPS-induced [3H]thymidine uptake by splenic lymphocytes, but its activity was less than 1/10 that of colistin. We conclude that colistin nonapeptide binds to LPS and possesses antiendotoxin activity. However, the anti-endotoxin activity of the nonapeptide is considerably less than that of its parent compound, colistin.

Enhancement of endotoxicity and reactivity with carbocyanine dye by sonication of lipopolysaccharide.

The specificity of endotoxin (lipopolysaccharide, LPS) in the carbocyanine dye reaction was investigated, and then a stoichiometric study of the dye-LPS interaction was conducted with attention to the relationship of biological activities of LPS to the reactivity with the dye. Absorption maxima of some bacterial components in the dye reaction were as follows; LPS from both Escherichia coli and Pseudomonas aeruginosa and lipid A from E. coli LPS, 465 nm; Shigella flexneri LPS, 460 nm; Salmonella minnesota R595 glycolipid, 470 nm; polysaccharide from E. coli LPS, 650 nm; yeast RNA, 620 nm; streptococcal M protein and pyrogenic exotoxin, 610 nm; and free fatty acids, 445-450 nm. The absorbance at 465 nm was increased approximately threefold by sonicating LPS for 1-3 min, which roughly paralleled the decrease in turbidity of the LPS aqueous solution. The Limulus amoebocyte lysate (LAL) gelation activity of LPS increased 10-fold when LPS was sonicated for 0.5-5 min, but it decreased to the control level after further treatment. This decrease, however, was overcome by sonication in the presence of 5 mmol of L-ascorbic acid used as an antioxidant. The LAL gelation activity of LPS was inactivated in parallel with an increase in the ratio (w/w) of dye to LPS from 1.73 to 6.90 in the dye-LPS mixture. Pyrogenicity of LPS was also clearly inactivated when the ratio was over 1.73. The ratios of the height of the beta band at 465 nm (dye-LPS complex) to that of the alpha band at 510 nm (free dye) were increased by sonicating LPS, indicating that the binding character, or stacking tendency, was increased by sonicating LPS.

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.

Involvement of central action of lipopolysaccharide in pyrogen fever.

To elucidate the mechanisms of fever response by bacterial pyrogen (lipopolysaccharide, LPS), we investigated both pyrogenicity and Limulus amoebocyte lysate (LAL) gelation activity of cerebrospinal fluid (CSF) withdrawn from febrile rabbits induced by i.v. injection of LPS. One ml of CSF was withdrawn from donor animals 2 hr after i.v. injection of E. coli LPS at graded doses of O, 1, 10 and 50 micrograms/kg, and its pyrogenicity was checked by administration into cisterna magna of recipient animals. Pyrogenicity was revealed only in the CSF withdrawn from the group injected with 50 micrograms/kg of LPS. Differences of both protein content and concentrations of ions (Na, K and Ca) were not obtained among the CSF withdrawn from control and LPS-injected groups. All of the above CSF had no LAL gelation activity, but the activity could be detected in the CSF withdrawn from animals injected with a higher dose of LPS (500 micrograms/kg, i.v.). LAL gelation activity of LPS dissolved in normal CSF was 2 orders less potent in comparison with that of LPS dissolved in saline, which suggested the presence of inhibitor(s) in CSF for LAL assay. Pyrogenicity was not revealed in the CSF withdrawn from hyperthermic rabbits induced by administration of reserpine after pretreatment with a monoamine oxidase inhibitor. These findings suggest that the central action of LPS is involved in pyrogen fever and that monoamines are not closely related to pyrogen fever.

Involvement of Central Action of Lipopolysaccharide in Pyrogen Fever

To elucidate the mechanisms of fever response by bacterial pyrogen (lipopolysaccharide, LPS), we investigated both pyrogenicity and Limulus amoe-bocyte lysate (LAL) gelation activity of cerebrospinal fluid (CSF) withdrawn from febrile rabbits induced by i.v. injection of LPS. One ml of CSF was withdrawn from donor animals 2 hr after i.v. injection of E. coli LPS at graded doses of 0, 1,10 and 50 μg/kg, and its pyrogenicity was checked by administration into cisterna magna of recipient animals. Pyrogenicity was revealed only in the CSF withdrawn from the group injected with 50 μg/kg of LPS. Differences of both protein content and concentrations of ions (Na, K and Ca) were not obtained among the CSF withdrawn from control and LPS-injected groups. All of the above CSF had no LAL gelation activity, but the activity could be detected in the CSF withdrawn from animals injected with a higher dose of LPS (500 μg/kg, i.v.). LAL gelation activity of LPS dissolved in normal CSF was 2 orders less potent in comparison with that of LPS dissolved in saline, which suggested the presence of inhibitor(s) in CSF for LAL assay. Pyrogenicity was not revealed in the CSF withdrawn from hyperthermic rabbits induced by administration of reserpine after pretreatment with a monoamine oxidase inhibitor. These findings suggest that the central action of LPS is involved in pyrogen fever and that monoamines are not closely related to pyrogen fever.

Endotoxaemia in Vibrio El Tor cholera

Endotoxaemia and endotoxin-induced changes were sought in Nigerian patients presenting with cholera/diarrhoea. The organism was Vibrio cholerae, bio-type El Tor, serotype Hikojima. The limulus amoebocyte lysate gelation test was used qualitatively by the clot method, whilst a spectrophotometric method was used quantitatively to measure endotoxin levels. 25 acutely ill patients tested had detectable endotoxaemia by the Escherichia coli endotoxin standard. The highest endotoxin level was found in a patient with sub-conjunctival haemorrhage. Changes in platelet counts, the detection of complement breakdown product C3d in plasma, the elevation of fibrin degradation products, the finding of elevated, normal or depressed C3 levels and the absence of circulating immune complexes, suggest a pathogenic role for endotoxin in Vibrio cholerae El Tor diarrhoea.

Production and properties of cyanobacterial endotoxins.

Lipopolysaccharides (LPS) were isolated from four species of cyanobacteria (Anabaena flos-aquae UTEX 1444. A. cylindrica, Oscillatoria tenuis, and O. brevis) frequently occurring in drinking-water supplies. The cyanobacterial LPS contained glucose, xylose, mannose, and rhamnose, but differed from the LPS derived from most gram-negative bacteria because of the variable presence of 2-keto-3-deoxyoctonate, heptose, galactose, and glucosamine. Cyanobacterial lipid A is characterized by long-chain saturated an unsaturated fatty acids and hydroxy fatty acids which show great diversity. Unlike lipid A from heterotrophic gram-negative bacteria, lipid A from cyanobacteria usually lacks phosphates. The detection of distinct O-antigen chemotypes indicates that LPS may be used for taxonomic classification. Isolated cyanobacterial LPS always induced Limulus amoebocyte lysate gelation. A. flos-aquae LPS gave a positive Schwartzman reaction. Endotoxins from A. cylindrica and O. brevis were toxic to mice when injected intraperitoneally. The cyanobacterial endotoxins showed generally lower biological activity than did LPS derived from common heterotrophic gram-negative bacteria. Nevertheless, cyanobacteria in algal blooms may be a significant source of endotoxins in water supplies.

Rapid detection of Gram-negative bacteriuria by Limulus amoebocyte lysate assay.

The Limulus amoebocyte lysate (LAL) test was evaluated for rapid detection of gram-negative bacteriuria in an adult patient population. Time to gelation of a standard LAL preparation was used as a measure of significant (greater than 10(5) bacteria per ml) gram-negative bacteriuria, and the results of 190 LAL assays were compared with quantitative urine cultures. Initially, 33 of 36 urine specimens containing greater than 10(5) gram-negative bacteria per ml were detected by LAL assay. The three false-negative LAL tests were the result of urine pH levels below the pH minimum for LAL gelation; neutralization of these urine specimens resulted in positive LAL assays and 100% correlation with culture results. All 36 bacteriuric urine specimens were LAL positive within 15 min, with the majority of assays (86.1%) being positive after only 10 min of incubation at 37 degrees C. These data compared favorably with gelation times of 15 min when 1 X 10(5) to 2 X 10(5) gram-negative bacteria per ml were added to sterile urine. Two urine samples obtained from male patients with culture-proven gonococcal urethritis yielded positive LAL assays. The LAL assay was shown to correctly differentiate 96.2% of urine specimens as containing less than 10(5) or greater than 10(5) gram-negative bacteria per ml. The results of this study have shown that the LAL test can be used as a rapid, simple, and reliable screening procedure for the diagnosis of clinically significant gram-negative bacteriuria.

Endotoxins, algae and Limulus amoebocyte lysate test in drinking water

Field and laboratory studies were conducted to determine the distribution of algae and bacteria, and investigate sources of endotoxins (lipopolysaccharides) in drinking water. The field survey was performed on five drinking water systems located in Allegheny County, Pennsylvania during the spring and summer of 1978. The highest concentrations of phytoplankton were found in uncovered finished water reservoirs. The major source of “endotoxic” response as measured by the Limulus amoebocyte lysate (LAL) gelation test was a non-specific reaction caused by algae. This was documented by a highly positive correlation of phytoplankton concentrations occurring in the reservoirs with respective LAL titers. Chlorella vulgaris (Chlorophyta) was the most common alga, whereas Schizothrix calcicola was the most dominant Cyanobacterium found in the five water systems. LAL gelation test with C. vulgaris grown in the laboratory verified the phenomenon observed on samples collected in the field and indicated a non-specific reaction, whereas S. calcicola cultures under identical conditions produced a specific response. Alkali and lysozyme treatments were successful in distinguishing specific and non-specific LAL reactions. These two techniques in conjunction with LAL test are recommended for drinking water quality assessment.