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.     

Positive Limulus amoebocyte lysate reactions with polyriboinosinic acid x polyribocytidylic acid.

The annealed copolymer polyriboinosinic acid x polyriboinosinic acid reacted with Limulus amoebocyte lysate to cause gelation at a concentration approximately 2,000-fold or greater than bacterial endotoxins. This copolymer was pyrogenic in rabbits and demonstrated hypochromicity, but no significant correlation was noted among Limulus amoebocyte lysate reactivity, progenicity, and hypochromicity. Like endotoxin, polyriboinosinic acid x polyribocytidylic acid did not react with purified Limulus coagulogen. Similar concentrations of the homopolymers polyriboinosinic acid and polyribocytidylic acid were negative or significantly below the Limulus amoebocyte lysate reactivity of the copolymear and essentially nonpyrogenic. Thus, polyriboinosinic acid x polyribocytidylic acid is a compound in addition to endotoxin that effects a positive Limulus amoebocyte lysate test.     

Survey for positive Limulus amoebocyte lysate test in plasma from humans and common research animals.

A survey for positive Limulus amoebocyte lysate tests was conducted on apparently healthy humans, mongrel dogs, rats, mice, rabbits, and squirrel monkeys. Only mongrel dog (45.8%) and human (32.8%) plasma samples gave positive tests. In dogs, a significant correlation between positive Limulus amoebocyte lysate tests and the presence of intestinal parasites was found. Positives found in human plasma samples were thought to be due to the presence of background levels of endotoxin or some possible mimicker substance found in the plasma after chloroform extraction. It was concluded that there was a need to distinguish between these positive Limulus tests and those which represent significant endotoxemia.     

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.     

Comparison of plasma extraction techniques in preparation of samples for endotoxin testing by the Limulus amoebocyte lysate test.

Due to the presence of inhibitory and possible mimicking substances in plasma difficulties have occurred in the use of the Limulus amoebocyte lysate test. Currently, there are a variety of extraction techniques discussed in the literature which are used to remove these interfering substances, but there is little information comparing these techniques. Five such procedures were compared in their ability to provide an extracted plasma sample in which low levels of endotoxin could be detected by the Limulus amoebocyte lysate test. Results indicated that some procedures adversely affected endotoxin detection. The dilution + heating extraction method was found to be as effective as the widely used chloroform extraction method. Comparison of Limulus amoebocyte lysate test results from healthy human plasma samples extracted by these two methods indicated that lysate type and not extraction procedure was associated with previously reported questionable positive tests. Thus, ambiguities associated with Limulus amoebocyte lysate tests of plasma samples may be due not only to extraction method but also the lysate type employed.     

Comparison of the histamine hypersensitivity and the Limulus amoebocyte lysate tests for endotoxin activity.

The histamine hypersensitivity test and the Limulus amoebocyte lysate test were compared for their effectiveness to quantitate endotoxin activity. The two tests compared favorably in all the trials, except with a sample of endotoxin from Brucella abortus that gave a positive Limulus amoebocyte lysate test at a concentration of 0.001 microgram, while failing to sensitize mice to histamine at a dose of 16 microgram per mouse. The Limulus amoebocyte lysate test was more sensitive than the histamine hypersensitivity test.     

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.     

Application of a Limulus test device in rapid evaluation of gonococcal and nongonococcal urethritis in males

A test device incorporating Limulus amoebocyte lysate (Mallinckrodt, Inc., St. Louis, Mo.) was developed for the rapid, presumptive diagnosis of gonococcal and nongonococcal disease in males. The device, which was evaluated in 550 men with exudative urethritis, consisted of a specimen collection syringe, a dilution reservoir containing 10 ml of pyrogen-free water, and a Limulus amoebocyte lysate single-test vial. After specimen collection, the syringe was affixed to the dilution reservoir for rapid, accurate dilution of the clinical sample. Contamination of the specimen and potential biohazards to the user were prevented. The diluted sample was then transferred (via the collection syringe) to the lysate test vial for assay of endotoxin. Various incubation times at 37 degrees C were also studied in an additional 301 male patients, and time was reduced from the standard 60 to 30 min while still retaining equivalent predictability of culture results (P less than 0.05). Of the 550 males evaluated with the test device, 366 had positive cultures for Neisseria gonorrhoeae, and 184 were negative. A sensitivity of 99.2% and a specificity of 96.7% were obtained with the test device. Overall ability to predict culture results was 98.4%. Gram-stain sensitivity and specificity were 96.4% and 99.5%, respectively, with an overall accuracy of 97.5%. There were no statistical differences between the Limulus amoebocyte lysate test and Gram stain in predicting cultures (P less than 0.05). Thus, use of the Limulus amoebocyte lysate test device would enable the private physician to make an accurate, presumptive diagnosis of gonococcal and nongonococcal disease in males with exudative urethritis within 30 min without the need of a microscope and to initiate proper therapy during the patient's initial evaluation.     

Endotoxin contamination of enzyme conjugates used in enzyme-linked immunosorbent assays.

The specificity of the enzyme-linked immunosorbent assay(s) is thought to depend on the specificity of the antibody used in the assay system. Therefore, the association of broadly reactive antigens like endotoxin with enzyme conjugates or other enzyme-linked immunosorbent assay reagents has the potential of altering the specificity of reactions in the enzyme-linked immunosorbent assay. Using the Limulus amoebocyte lysate assay, we demonstrated that commercially prepared conjugates of goat anti-human immunoglobulin G peroxidase, goat anti-rabbit immunoglobulin G alkaline phosphatase, rabbit anti-human immunoglobulin G, and other enzyme conjugates contained endotoxin. Furthermore, the staphylococcal protein A, horseradish peroxidase, and bovine alkaline phosphatase used to prepare enzyme conjugates also contained endotoxin. Commercially obtained bovine alkaline phosphatase contained as much as 1.0 microgram of endotoxin per ml of enzyme solution. Both commercially prepared enzyme conjugates and those prepared by us contained endotoxin as determined by their absorption to immobilized monoclonal antibody to lipid A or to immobilized Limulus amoebocyte lysate. The results of this study further suggest that the endotoxin was associated with the enzyme component of the conjugate. In a competitive inhibition enzyme immunoassay, 10 micrograms of lipid A per ml inhibited binding of the enzyme conjugate to adsorbed Limulus amoebocyte lysate, thereby confirming that endotoxin mediated the binding of the conjugate in that system. The potential significance of endotoxin bound to enzyme conjugates may be far reaching because of the ubiquity of endotoxin in conjugates and the prevalence of antibodies to endotoxin in mammalian serum.     

Detection of endotoxin in the plasma of patients with gram-negative bacterial sepsis by the Limulus amoebocyte lysate assay.

A total of 120 Limulus amoebocyte lysate (LAL) determinations were made on plasma obtained from normal, healthy human blood donors. Results demonstrated a mean endotoxin level in blood of 0.02 to 1.57 pg/ml. The amount of Escherichia coli endotoxin added to human plasma samples can be quantitated by both nephelometry and turbidimetry. Endotoxin-spiked samples were shown to be significantly different from unspiked samples. When plasma samples were collected from 45 patients hospitalized at three centers, a strong association was demonstrated between a positive Limulus amoebocyte lysate assay and a septic condition. Sensitivity, specificity, and false-positive and false-negative rates for the Limulus amoebocyte lysate assay as a diagnostic test for gram-negative bacteremia were estimated.     

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 the lipid. Using this approach (buffer method), we observed that less than 10% of Salmonella minnesota smooth LPS is incorporated into multilamellar 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.     

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.     

Clinical value of endotoxin determination in infection. Comparison of the Limulus amebocyte lysate test with detection of bacterial pathogens

To evaluate usefulness of Limulus amoebocyte lysate test and blood culture in the diagnosis of septicemia both tests were performed in 27 intensive care patients. Test results were compared with a clinical sepsis score. Ten (62%) out of 16 patients with clinical diagnosis of septicemia showed a positive endotoxin test and 11 (69%) a positive blood culture. In 14 patients (87%) either endotoxin test or blood culture revealed a positive result. Two out of 11 patients (20%) classified by the sepsis score as non-septic showed positive blood cultures as well as positive endotoxin tests. 4 patients with gram-positive bacteria in the blood cultures showed a positive endotoxin test. Due to lack of sensitivity and specificity the Limulus amoebocyte lysate test is of rather low value in the diagnosis of septicemia. Simultaneous performance of Limulus amoebocyte lysate test and blood culture is able to improve the sensitivity, which then over-rules the one obtained when only blood cultures are performed.     

Rapid diagnosis of gram-negative bacterial meningitis by the Limulus endotoxin assay.

The Limulus amoebocyte lysate endotoxin assay was evaluated as a method for rapid diagnosis of acute bacterial meningitis in a series of 305 patients. The results of Limulus assays on cerebrospinal fluid (CSF) samples from these patients were compared with the results for each patient of routine bacterial cultures and Gram stains. Positive Limulus tests were obtained on initial CSF specimens from 84% of patients with culture-proven bacterial meningitis, including all patients with meningitis due to gram-negative organisms. Initial Gram-stained smears revealed the presence of organisms in 68% of the patients. One patient with pneumococcal meningitis had a weakly positive Limulus assay, whereas patients with meningitis due to other gram-positive organisms, those with aseptic meningitis, or patients without meningitis had negative CSF Limulus tests. The Limulus assay also demonstrated the persistence of endotoxin in the CSF of certain patients during antibiotic therapy, especially patients with Haemophilus influenzae meningitis. The Limulus test proved to be a rapid, reliable indicator of the presence of gram-negative organisms in the CSF of patients suspected of acute bacterial meningitis.     

Differential induction of tumor necrosis factor by bacteria expressing rough and smooth lipopolysaccharide phenotypes.

The lipid A portion of the lipopolysaccharide (LPS) molecule of gram-negative bacteria has the ability to turn on the production of tumor necrosis factor (TNF) in macrophage cells. The question addressed in this paper was whether the presence of the polysaccharide moiety on the LPS molecule had any bearing on this ability. The question was asked (i) by using isolated LPS from a series of Salmonella mutants having progressively less polysaccharide attached to the lipid A portion of the molecule and (ii) by using whole bacteria expressing alternatively the smooth or rough LPS phenotype. Isolated LPS and bacteria were examined for their abilities to induce bioactive TNF in the mouse macrophage cell line RAW 264.7. The results indicated that the presence of long- or short-chain polysaccharide moieties had no bearing on the ability of the isolated LPS molecule to induce TNF. However, the presence of long-chain polysaccharides attached to the lipid moiety on the intact smooth bacterium was associated with a decreased ability to induce TNF. To test whether the bacteria were inducing TNF by a cell (bacterium)-to-cell (macrophage) contact mechanism or through a releasable product, the bacteria were removed from direct contact with the macrophage cells by using a Transwell filter insert. Under these conditions the rough bacteria continued to induce TNF, while the smooth bacteria were no longer capable of doing so. When filtrates from the bacteria were examined in the Limulus amebocyte lysate assay, the results showed that the rough bacteria were releasing approximately a log order more Limulus amebocyte lysate activity than the smooth bacteria. The results of this study suggest that rough bacteria may be superior inducers of TNF compared with their smooth counterparts because of a greater propensity to shed their LPS.     

Preparation and characterization of a nontoxic polysaccharide-protein conjugate that induces active immunity and passively protective antibody against Pseudomonas aeruginosa immunotype 1 in mice.

Acid treatment of Pseudomonas aeruginosa immunotype 1 lipopolysaccharide generated a low-molecular-weight polysaccharide fraction that was detectable in agar gel immunodiffusion but did not induce antibodies or resistance to infection in mice. The polysaccharide was treated with periodate to generate additional aldehyde groups. Oxidized polysaccharide was covalently coupled by reductive amination to 1,4-diaminobutyl-derivatized bovine serum albumin. Physical properties of the conjugate were characterized by gel filtration and high-pressure liquid chromatography. The gelation activity of the conjugate in the Limulus amoebocyte lysate assay was 4,000-fold less than native lipopolysaccharide by weight. Mice immunized with the conjugate resisted challenge with P. aeruginosa immunotype 1 that killed 90% of mice immunized with saline. Immunization with the conjugate vaccine induced humoral immunoglobulin G that passively protected normal and burned mice. These results indicate that conjugation of nonimmunogenic polysaccharide antigen of P. aeruginosa restores immunogenicity similar to that of native lipopolysaccharide without restoring endotoxicity inherent in lipopolysaccharide.     

The role of amoebocytes in endotoxin-mediated coagulation in the innate immunity of Achatina fulica snails

Achatina amoebocyte lysate (AAL) derived from amoebocytes of Achatina fulica was activated by Gram-negative bacterial endotoxins in a time-dependent manner resulting in gel formation/coagulation. The activation and maximum proliferation of amoebocytes was observed 40 min after intramuscular injection (20 microg/snail) of endotoxin. Endotoxin-mediated proteolytic activity of AAL towards a serine-protease-specific chromogenic substrate was maximum at pH 8.0, 37 degrees C and within 15 min in a divalent-cation-dependent manner. The AAL activity induced by the endotoxin was directly dependent on the endotoxin concentration, showed a high specificity and saturated at higher endotoxin concentrations. An endotoxin-sensitive factor (ESF) was purified from AAL to apparent homogeneity by single-step affinity chromatography on a heparin-Sepharose 4B column. Native ESF of molecular weight 140 000 was composed of two identical subunits of molecular weight 70 000 attached through non-covalent association. A strong binding to endotoxin (Escherichia coli 055:B5) was exhibited by ESF with a 40-fold higher biological activity than AAL. The ESF was shown to have a unique Phe-Ile active site with regard to its alternate activation by alpha-chymotrypsin instead of endotoxin. The ESF was characterized as a serine protease type as evidenced by potent inhibition with specific inhibitors.     

Augmentation of endotoxin fever by recombinant human beta interferon in rabbits.

Nonpyrogenic amounts of endotoxin (0.1 to 1 ng/kg), hardly detectable by conventional Limulus amoebocyte lysate tests, could produce a fever of around 1 degree C when injected with a nonpyrogenic dose (6 X 10(5) U/kg) of recombinant human beta interferon (IFN-beta) in rabbits. Release of endogenous IFN and tumor necrosis factor by endotoxin was also dramatically increased by recombinant human IFN-beta, and their levels in the blood were closely correlated with the increase of body temperature. These data suggest, if the synergism between IFN and endotoxin also operates in the homologous system (human IFN-human cells), that contaminating endotoxin in IFNs, even if not detectable by Limulus amoebocyte lysate test, can contribute to IFN fever to a considerable extent in humans.     

Non-standard biological activities of lipopolysaccharide from Helicobacter pylori

As assessed by the lipopolysaccharide (LPS)-specific chromogenic Limulus amoebocyte lysate (LAL) assay, Helicobacter pylori LPS extracted by the phenol-water procedure showed full potency to coagulate LAL, as did LPS from Salmonella minnesota and Escherichia coli. However, pretreatment of H. pylori LPS with polymyxin B, which easily destroys the endotoxic activity of enterobacterial LPS/lipid A, had little effect on the LAL coagulation activity, although the same treatment of E. coli LPS markedly diminished its activity. The H. pylori LPS induced very weak production of nitric oxide (NO) or tumour necrosis factor (TNF) by murine macrophages and TNF by human peripheral whole blood in vitro in comparison with S. minnesota LPS. These findings indicate that H. pylori LPS has the unique endotoxic characteristic of retaining full LAL coagulation activity with polymyxin B resistance, despite losing its endotoxic potencies such as the ability to induce NO and TNF production.     

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.