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PubMed1994

Quantitative assay of (1-3)-beta-D-glucan in culture media of Candida albicans using the G-test.

The diagnosis of invasive candidiasis is often difficult. The limulus test, which has been used for the assay of endotoxin in blood, was also found to react with (1-3)-beta-D-glucan, a component of the fungal cell wall. The factor in the limulus test that is activated by glucan, but not by endotoxin, is called factor G. The G-test utilizes the activation pathway starting with factor G, and sensitivity reacts with trace amounts of glucan. In this study, we investigated in vitro proliferation of Candida albicans and changes in the glucan concentration of RPMI-1640 media in the presence and absence of neutrophils and antifungal agents, as a pilot evaluation to possible clinical applications of the G-test. The proliferation of C. albicans and the glucan level measured in the culture media showed parallel changes. The glucan level in the culture media also increased when C. albicans was phagocytosed and digested by neutrophils, but not with administration of amphotericin B. The G-test closely reflected quantitative changes of C. albicans in vitro, and should be considered for future clinical studies in the diagnosis and evaluation of therapeutics in invasive candidiasis.

Amphotericin B↗
PubMed1976

Limulus amebocyte lysate test in neonates.

The limulus amebocyte lysate test for endotoxin was done on samples of blood from 22 well babies and 33 neonates in an intensive-care nursery. The objective was to determine whether falsely positive test results occurred in samples from newborn infants during acquisition of usual bowel flora. One neonate had a transiently positive limulus amebocyte lysate test; he had clinical signs of spesis, but no bacteremia could be documented. Unlike the nitroblue tetrazolium test, the limulus amebocyte lysate test does not appear to be regularly positive in neonates. The sensitivity of the test in detection of endotoxemia associated with gram-negative spesis in the neonate remains to be determined in a large prospective study.

Arthropods↗
PubMedDate not supplied

Identification and characterization of lipopolysaccharide in acetic acid bacteria.

BACKGROUND: Lipopolysaccharide (LPS), a major component of the cell walls of Gram-negative bacteria, was one of the main components of Coley's vaccine and is known to have strong adjuvanticity. Though it is known that LPS exists in the digestive tract of organisms, the biological significance for the organism has not been clarified. In this study, the correlation between the structure and function of LPS was determined using acetic acid bacteria. These are Gram-negative bacteria consumed in human diets. MATERIALS AND METHODS: Extracts were obtained from a strain of acetic acid bacteria which is used for producing vinegar. Determination of the LPS neutralizing activity was carried out by the Limulus test. Tumor necrosis factor (TNF) and nitric oxide (NO) production were then observed after the addition of the extracts to murine monocyte macrophages (RAW264. 7), with or without Polymyxin B. TNF production in peritoneal macrophages derived from LPS-low responsive mice (C3H/HeJ) was studied after the addition of extracts. RESULTS: The extracts were shown to be positive only in LPS-specific Limulus test and were negative in the (1,3)-beta-D-glucan-specific Limulus test. Both extracts induced NO and TNF production in RAW264. 7 cells, but this was inhibited by the presence of Polymyxin B. TNF production was inhibited in peritoneal macrophages from LPS low-responsive mice (C3H/HeJ). CONCLUSION: LPS with macrophage-activating activity is present in acetic acid bacteria, routinely consumed by humans.

Acetobacter↗
PubMed1992

Environmental endotoxin measurement: the Kinetic Limulus Assay with Resistant-parallel-line Estimation.

A Limulus assay method was specifically designed for environmental endotoxin aerosols. Application of new statistical and sample preparation methods strengthened the validity and precision of the Limulus test. Statistically, the Kinetic Limulus Assay with Resistant-parallel-line Estimation (KLARE) differed from conventional analytic methods (as used in chromogenic assays and other kinetic methods) by routinely using a dilution series of the unknown sample as well as the standard to compute potency and an estimate of variance for each sample. Analysis of dose-response slopes for the standard and unknowns detected inhibition and enhancement effects--without multiple assay. Concentration-dependent interference and a more complex, concentration-independent interference with the Limulus assay were detected. Resistant regression and a standardized data analysis corrected for concentration-dependent interference. Sample preparation in a buffer eliminated concentration-independent interference and, thus, improved both the validity and the precision of potency measurements. The utility of a sample buffer and of parallel-line analysis, with both turbidimetric and chromogenic lysates, was demonstrated by assay of three control standard LPS and reference LPS (EC5). The limit of detection for endotoxin was less than 1 pg/ml in buffer. Samples containing greater than or equal to 10 pg/ml were measured with a coefficient of variation of approximately 6% in a single assay. Reproducibility of potency estimates for four samples over 3 days was compared on the basis of standard errors of the mean. The conventional method gave on average a CV of 65% while the resistant-parallel-line method gave, on average, a CV of 6%. Also, the conventional method failed to detect interference and, thus, included data from invalid assays. Conventional analysis of environmental aerosol samples was highly sensitive to the choice of dilution factor causing as much as 1000% variation in the result. By contrast, KLARE results changed by at most 30% with similar changes in initial dilution because KLARE was able to detect, and correct for, the influence of interferant compounds.

Aerosols↗
PubMed1979

Limulus lysate assay for early detection of certain Gram-negative corneal infections.

The limulus endotoxin assay has been previously demonstrated to be the most sensitive method available for detection of bacterial endotoxin. A commercially available form of limulus amoebocyte lysate was used in this study for detection of Gram-negative corneal infections in both experimental animals and in a group of nine patients. The limulus assay enabled rapid detection of Gram-negative infections in both the experimentally induced ulcers in rabbits and in the patients studied. False-positive reactions did not occur in corneal infections due to either Gram-positive bacteria, fungi, or herpes simplex keratitis. The limulus test proved to be more sensitive than examination of Gram-stained smears of corneal scrapings and became positive earlier than bacterial cultures. The limulus test was helpful in the diagnosis of partially antibiotic-treated corneal infections but could not be used to assess the response to antimicrobial therapy, since endotoxin persisted in the corneal scrapings for some time after initiation of therapy.

Animals↗
PubMed1978

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.

Animals↗
PubMedDate not supplied

Effect of phenothiazines, benzo[a] phenothiazines, benz[c]acridines and Pentaglobin on endotoxin.

The endotoxin neutralizing effects of several phenothiazines, benzophenothiazines and Pentaglobin (control) were investigated by spectrophotometry, tumor necrosis factor (TNF) induction and the conventional Limulus test. In animal experiments, some beneficial effects of complex forming compounds were found, however, the compounds could not completely inactivate the biological effect of endotoxin in the Limulus test. The complex formation between endotoxin and the compounds were revealed in spectrophotometry. The TNF inducing effect of compound-endotoxin complexes was markedly reduced by some phenothiazines and benzo[a]phenothiazines in leukocytes. Benz[c]acridines and Pentaglobin could not neutralize completely the TNF induction of E. coli endotoxin. The recent findings indicate that multifocal effects of phenothiazines and benzophenothiazines can be responsible for anti-endotoxin action in vivo. Hypotensive action in experimental animals was reduced by some phenothiazines in some preliminary experiments.

Acridines↗
PubMed1977

Gelation of Limulus lysate by synthetic dextran derivatives.

The Limulus test has been considered specific for the presence of bacterial endotoxins. To synthesize a simple model of endotoxin, palmitoyldextran phosphate was prepared by modification of dextran by palmitoylation and phosphorylation. The present studies indicated that a variety of polysaccharide derivatives, such as palmitoyldextran phosphate, palmitoyldextran, and dextran phosphate, give a positive Limulus test and show pyrogenic activity, except for low molecular dextran derivatives. On the other hand, polysaccharides, such as dextran, starch (soluble-, chitosan, xylan, and lentinan, were negative in these assays. The gelation reaction of Limulus lysate by modified dextran derivatives may depend on the molecular weight or modification of polysaccharides by palmitoylation and/or phosphorylation to a great extent.

Chemical Phenomena↗
PubMed1987

Presence of endotoxemia and its relationship to liver dysfunction in patients with typhoid fever.

Twenty-one patients with typhoid fever were studied to evaluate the presence of endotoxin in peripheral blood and its relationship to the incidence and features of hepatic dysfunction which may occur during this disease. The limulus test for endotoxin was positive in the plasma samples of all patients prior to treatment. Liver dysfunction, as assessed by fasting and postprandial serum bile acid levels and by standard biochemical tests, occurred in 90% of patients. In seven, the injury was purely cholestatic (elevation of postprandial serum bile acid levels, alone); in 12, it was of mixed cholestatic-hepatocellular type (elevation of both serum bile acids and aminotransferase levels). After recovering, the limulus test was negative and liver function tests returned to normal values in all patients. The results demonstrate that endotoxemia is present in patients with typhoid fever. In addition, since endotoxin can impair bile secretion, our results suggest that endotoxin may have a pathogenetic role in the development of liver injury during typhoid fever.

Adolescent↗
PubMed1996

Detection of gram-negative bacteraemia in early sepsis by a quantitative chromogenic and kinetic endotoxin assay. The Study Group.

A kinetic chromogenic limulus test was carried out in order to investigate the possibility of a sensitive and specific detection of circulating endotoxin during the first 24 h of septic shock or severe sepsis in 76 patients. Two commercial kits, Whittaeker (W) and Chromogenix (C), were used. Blood culture was taken as a reference. At 1:10 plasma dilution (a currently used dilution in the end point limulus test) abnormal reaction kinetics were found in 13% and 41% of tests, for C and W respectively (P = 0.0008), resulting in unreliable results. Retesting plasma at a greater dilution, until the reaction kinetic was identical to calibration curve control values, gave similar results between the two kits and a better accuracy. Beyond a 0.5 EU mL-1 endotoxin level, the probability of Gram-negative bacteraemia was high (sensitivity = 0.53 and 0.47; specificity = 0.95 and 0.93 for C and W respectively). This kinetic limulus amoebocyte lysate (LAL) test may be useful in therapeutic decisions for treatment of endotoxaemia.

Aged↗
PubMed1986

Low endotoxic activities of synthetic Salmonella-type lipid A with an additional acyloxyacyl group on the 2-amino group of beta (1-6) glucosamine disaccharide 1,4'-bisphosphate.

A synthetic lipid A (Salmonella type, compound 516), beta (1-6)-linked D-glucosamine disaccharide 1,4'-bisphosphate, with three acyloxyacyl groups and one hydroxyacyl group, i.e., (R)-3-hexadecanoyloxytetradecanoyl, (R)-3-hydroxytetradecanoyl, (R)-3-dodecanoyloxytetradecanoyl, and (R)-3-tetradecanoyloxytetradecanoyl groups at the 2-amino, 3-hydroxyl, 2'-amino, and 3'-hydroxyl groups, respectively, was less biologically active than the synthetic Escherichia coli-type lipid A (compound 506), which has only two acyloxyacyl groups at the 2' and 3' positions and is substituted with a (R)-3-hydroxytetradecanoyl group at the 2-amino group. Compound 516 exhibited considerably weaker pyrogenic and leukopenic activity than compound 506, and it scarcely prepared rabbit skin for the Shwartzman reaction and lacked lethal toxicity on chicken embryos, although its lethal toxicity in galactosamine-loaded mice was as strong as that of compound 506. Compound 516 was also less active than compound 506 or natural E. coli lipid A (from Restrain F515) in other biological test systems, such as the Limulus test, stimulation of macrophages and lymphocytes, and interferon-inducing activity but not for interleukin-1 induction or complement activation. This observation suggests that there is an optimal number of acyloxyacyl groups on the glucosamine backbone for producing the biological activities of lipid A, especially the endotoxic activities. The 4'-monophosphate analog (compound 514) of compound 516 in general had significantly weaker activity than compound 516 in the above assays, most probably because of its greater hydrophobicity and consequently lower solubility in assay systems. Bacterial R595 lipid A derived from S. minnesota Re-mutant, which is a mixture of compounds 516 and 506, their 4'-monophosphate analogs and other compounds, exerted intermediate degrees of activity between compounds 506 and 516 in the various test systems employed.

Adjuvants, Immunologic↗
PubMed1979

Control of endotoxinemia in liver disease by lactulose and paromomycin.

Through the use of the Limulus test research has been carried out on gram-negative endotoxin in patients with hepatic cirrhosis, chronic hepatitis, acute hepatitis, and in a control group. The positivity of this test in patients with cirrhosis and chronic hepatitis was 93.3% and in cases of acute hepatitis it was 90.9%. The effect of the combined administration of lactulose and paromomycin on endotoxin blood levels has been evaluated in a group of 9 patients with acute hepatitis, 8 with cirrhosis, 1 in hepatic coma, and 1 patient with chronic persistent hepatitis: in 18 of the 19 patients the Limulus test became negative. The results have been discussed in relation to clinical and laboratory data, and to recent data concerning the interaction between intestinal bacterial flora, endotoxin, and liver. Hypotheses have been proposed regarding the hepatocellular c-AMP mediated mechanism of endotoxin action.

Chronic Disease↗
PubMed1992

A new sensitive microplate assay of plasma endotoxin.

We have developed a microplate method for determining endotoxin in platelet-rich plasma-using Endospecy, an endotoxin-specific chromogenic Limulus test reagent. Nonspecific activators and inhibitors of the test were eliminated by exposing samples (5 microliters) to the alkali reagent consisting of KOH, CaCl2, Triton X-100, ethyleniminepolymer and N,N-bis(2-hydroxyethyl)glycine. The recoveries of various endotoxins were almost complete and not enhanced by dilution. The dose-response curve was linear over endotoxin concentrations of 2-400 pg/ml with good precision (C.V. less than 5.0%). Normal human plasmas (n = 30) contained less than 5.0 pg/ml of endotoxin in reference to that of Escherichia coli 0111: B4. All plasma samples with high concentration of endotoxin by a conventional method showed high values by the microplate assay as well. Since it does not require centrifugation, the new treatment allows the whole reactions to proceed on the same microplate. This permits us to apply the Limulus test to an automated assay system, making plasma endotoxin determination simpler and more rapid than a conventional test tube method.

Detergents↗
PubMed1992

[Application of the limulus amebocyte lysate test to measurement of endotoxin in therapeutic human plasma protein fraction. Comparison with the rabbit pyrogen test].

We applied the limulus amebocyte lysate (LAL) test to the detection of bacterial endotoxins in therapeutic human plasma protein fraction (PPF) and compared the LAL-test with the rabbit pyrogen test. Two endotoxin-specific LAL-reagents were used for the colorimetric method and turbidimetric kinetic method. The amounts of added endotoxin to the PPF were correctly estimated by either method. The results of four independent assays for the 53 samples of PPF corresponded well with each other (correlation coefficient: 0.851-0.959, regression coefficient: 0.898-1.151). The amounts of endotoxin in the PPF estimated by the LAL-test significantly correlated with the rise of body temperature in rabbits (correlation coefficient: 0.547-0.642, and 0.911-0.934 for the endotoxin added samples). These results suggest that the LAL-test could be used as an alternative method for the rabbit pyrogen test to PPF.

Animals↗
PubMed1983

Incidence of endotoxemia in pigs with ischemic hepatic necrosis treated by hemodialysis. Prevention of endotoxemia with lactulose.

The incidences of endotoxemia and bacteremia were evaluated in 30 pigs with ischemic hepatic necrosis treated by hemodialytic procedures. Prior to induction of hepatic ischemia, ten pigs underwent bowel cleansing by means of an oral dose of magnesium sulfate, and 20 received a combination of magnesium sulfate and lactulose. Endotoxemia and bacteremia seldom occurred during the development of hepatic encephalopathy, but the incidence of both increased markedly shortly before death. Pigs pretreated with magnesium sulfate and lactulose however did not develop preterminal endotoxemia. A significant relation between endotoxemia or bacteremia and survival was not found, irrespective of pretreatment with lactulose. Of the positive limulus tests, 67% were accompanied by a positive blood culture, while 42% of all positive blood cultures were associated with a positive limulus test. Dialysis with dialysates contaminated with endotoxins did not increase the risk of endotoxemia. It is concluded that in an animal model of ischemic hepatic necrosis (1) endotoxemia and bacteremia appear mainly in the preterminal stage, but do not influence the duration of survival significantly; (2) lactulose prevents endotoxemia and (3) dialytic procedures do not increase the risk of endotoxemia and bacteremia.

Animals↗
PubMed1976

Yersinia pestis infection in Vietnam. II. Quantiative blood cultures and detection of endotoxin in the cerebrospinal fluid of patients with meningitis.

Quantitative blood cultures were obtained from 42 patients with acute Yersinia pestis infection to determine whether the concentration of bacteria in blood influenced the clinical severity and outcome of illness. In 17 bacteremic patients, colony counts in blood cultures ranged from less than 10 to 4 X 10(7)/ml. Three of five patients with colony counts of greater than 10(2)/ml died, and two patients survived episodes of hypotension. Results from plasma limulus tests were positive at the time of admission in three of 10 patients tested, and these three patients had bacteremia with colony counts of greater than 10(2)/ml. Meningitis developed in three patients and pneumonia in two patients; these five patients a-l had buboes in the axillary region. Endotoxin was detected with the limulus test in the cerebrospinal fluid in the three patients with meningitis. Ten patients randomly assigned to receive streptomycin or trimethoprim-sulfamethoxazole survived. Those treated with streptomycin had a shorter median duration of fever and a lower incidence of complications than did the patients treated with trimethoprim-sulfamethoxazole.

Adolescent↗
PubMed1978

Effects of certain cations (Fe, Zn, Mg, and Ca) on bacterial endotoxins.

The natural occurrence of cations Fe, Zn, Mg, and Ca in the lipopolysaccharide (LPS) of both the S and R forms of Shigella dysenteriae 1 was studied. LPS preparations were obtained either by phenol-water extraction (according to the method of Westphal et al., Z. Naturforsch. 7b:148-155, 1952) or by extraction of cells with hypertonic sodium chloride-sodium citrate (according to the method of Raynaud and Digeon, C. R. Acad. Sci. (Paris) 229:564-566, 1949), with subsequent chromatographic purification on Sephadex G200 and Sepharose 4B columns. The cation in highest concentration in the Westphal extract was Mg(2+) (as much as 30 mug/mg), and the lowest one was Fe (ca. 0.10 mug/mg). In LPS of the Raynaud type, the cation in highest concentration was Ca(2+) (as much as 13 mug/mg), and the lowest one was Fe (ca. 0.10 mug/mg). The effects of increasing and decreasing the concentrations of cations (Fe, Zn, Mg, Ca) upon the biological activity of the endotoxins was evaluated by using toxicity in mice and the Limulus test. It appeared that increased concentrations of Fe (chiefly of Fe(3+)) decreased the toxicity of the R form of LPS, whereas Mg(2+) decreased the toxicity of the S form. After prolonged dialysis of LPS preparations against deionized water, there was no consistent relationship between toxicity as determined in white mice and with the Limulus test.

Calcium↗