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PubMed1979

Tachypleus lysate test for endotoxin in patients with Gram negative bacterial infections.

Amoebocyte lysate from the horseshoe crabs (Tachypleus gigas) which abounds in the Gulf of Thailand was used to detect endotoxin in patients with Gram-negative bacteremia, in patients with Gram-positive bacterial infections as well as in the control. The Tachypleus lysate test (TLT) was positive in 94.4% of 36 patients with Gram-negative bacteremia before initiation of antibiotic therapy. Only 4% of 50 healthy individuals were positive and all 7 patients with Gram-positive bacterial infections were negative. The threshold sensitivity of TLT was 0.625 micrograms endotoxin per ml of the plasma. In comparison with the commercial Limulus lysate test (LLT), the TLT was slightly more sensitive in exhibiting higher grade of reaction, eventhough the threshold sensitivity was the same.

Endotoxins↗
PubMed1995

A rapid test for endotoxin in whole blood.

A rapid whole blood agglutination test has been developed for the detection of endotoxin. The test reagent consists of polymyxin B (PmB) conjugated to the Fab fragment of the anti-glycophorin antibody 1C3/86. After addition of reagent to whole blood, red cell agglutination occurs within two minutes in samples from endotoxaemic patients or with the addition of either whole Gram negative bacteria, supernatants from Gram negative bacterial cultures or purified endotoxin. In clinical samples there was a strong correlation (r = 0.83) between the strength of agglutination and the level of endotoxin measured by the Limulus amaebolysate test (LAL). The prospect of a rapid and accurate test for endotoxin may enable better clinical management of Gram negative sepsis.

Adult↗
PubMed1982

Immunoradiometric assay of lipid A: a test for detecting and quantitating endotoxins of various origins.

The ability to measure circulating endotoxin in various disease states has been hampered by the lack of a specific and quantitative assay. The test most commonly used has been the Limulus gelation assay, which measures an enzymatic effect of endotoxin rather than the substance itself. Based on a solid-phase immunoradiometric assay previously developed to detect the specific lipopolysaccharide from Escherichia coli 026, a similar assay has been developed for the lipid A moiety of endotoxins. The assay uses rabbit antibodies to lipid A which do not react with ketodeoxyoctonate, myristic or beta-hydroxymyristic acids, and detects lipid A obtained from endotoxins of various origins after acid hydrolysis of lipopolysaccharide. Experiments in rats given exogenous endotoxin suggest that this assay can be useful for quantitation of bacterial endotoxins in serum and for studying the pathophysiology of experimental endotoxemia.

Animals↗
PubMed2006

Endotoxin content in endodontically involved teeth. 1975.

Fluid was aspirated from the root canals of 40 endodontically involved teeth. This fluid was assayed for endotoxin with the limulus lysate test. Pulpless teeth contained greater concentrations of endotoxin than those with vital pulps. Symptomatic teeth also contained more endotoxin than asymptomatic teeth.

Body Fluids↗
PubMed1990

[Comparison of D-arabinitol, candida antigen, and beta-glucan, measured by the chromogenic limulus assay, in the rabbit model of systemic candidiasis].

In this study, we measured D-arabinitol, candida antigen and beta-glucan in the rabbit model of systemic candidiasis. It was recently reported that the factor G, one of the coagulation enzymes from the horseshoe crad amebocyte lysate, reacts with beta-glucan or polysaccharide from fungus, and not with endotoxin. The newly developed endotoxin specific chromogenic Limulus assay (endospecy test), which is devoid of factor G, did not react with any rabbit serum. But the beta-glucan measured by the conventional Limulus assay (toxicolor test), which contains the factor G, was elevated (from 10 pg/ml to 100 pg/ml) in all cases of rabbit model of system candidiasis. Serum D-arabinitol, measured by Sone's enzymatic fluorometric method, increased in all cases, but Cand-Tec (candida detection system by latex agglutination) showed only one positive case in five innoculated rabbits.

Animals↗
PubMed1976

Portal venous and systemic endotoxaemia in patients without liver disease and systemic endotoxaemia in patients with cirrhosis.

Systemic endotoxaemia without evidence of gram-negative bacterial infection occurs in liver diseases in man. The endotoxaemia is probably due to impaired hepatic clearance of endotoxin absorbed from the gastrointestinal tract, but portal venous endotoxaemia has never been reported in man. By means of the limulus gelation test, portal venous blood from 21 patients without parenchymal liver disease and arterial blood from 21 patients without parenchymal liver disease and 31 patients with cirrhosis was examined for endotoxin. Portal venous endotoxaemia was found in 9 of 21 samples and systemic endotoxaemia was found in 2 of 21 samples from patients without liver disease. Systemic endotoxaemia in cirrhosis occurred with a frequency of 15/31. No relationship to gram-negative bacteraemia was found. Leucocytosis was only seen in endotoxin-positive patients with cirrhosis. In cirrhosis higher levels of E. coli O antibodies were found in endotoxin-positive than in endotoxin-negative patients, supporting the view that the limulus gelation test specifically detects endotoxin (i.e. E. coli O antigen). The study suggests that endotoxin is a normal constituent of portal venous blood in man. The normal human liver clears endotoxin from the portal venous blood. This effect is diminished in cirrhosis, most probably owing to decreased phagocytic function of the liver. The increased humoral immune response in cirrhosis may be related to spillover of endotoxin from the liver.

Antibodies, Bacterial↗
PubMedDate not supplied

[A study of the Limulus amebocyte lysate-reactive substances flushed from cellulose hemodialysis membranes].

It is shown that substances washed from cellulose hemodialysis have no pyrogenic activity, react positively in Limulus-amebolysate test used for bacterial endotoxin assay. Dialyzers are compared by the yield of washed compounds, the importance of the dialyzer sterilization method is pointed out. The influence of chemical processing in repeated usage of dialyzers on the yield of the washed from the membranes compounds and time course of their washing in long-term cleaning of dialyzers and during hemodialysis have been outlined. The compounds were found active in relation to human complement system.

Adult↗
PubMed2005

Investigation into the interaction of the phosphoporin PhoE with outer membrane lipids: physicochemical characterization and biological activity.

Outer membrane pore proteins such as phosphoporin (PhoE) are important constituents of Gram-negative bacteria such as Escherichia coli. We have studied the interaction of PhoE with the membrane-forming lipids phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) from the inner and lipopolysaccharide (LPS) from the outer leaflet of the outer membrane. These investigations comprise functional aspects of the protein:lipid interaction corresponding to the outer membrane system as well as the activity of LPS:PhoE complexes in the infected host after release from the bacterial surface. The interaction of the lipids PE, PG, and LPS with PhoE was investigated by analysing molecular groups in the lipids originating from the apolar region (methylene groups), the interface groups (ester), and polar groups (phosphates) applying Fourier-transform infrared spectroscopy (FTIR), and by analysing the phase transition behaviour of the lipids using FTIR and differential scanning calorimetry (DSC). The activity of PhoE and LPS:PhoE complexes was investigated in biological test systems (human mononuclear cells and Limulus amebocyte lysate assay) and with phospholipid model membranes using fluorescence resonance energy transfer spectroscopy (FRET). The results show a strong influence of PhoE on the mobility of the lipids leading to a considerable fluidization of the acyl chains of LPS, but much less to those from phospholipids: PhoE released from the outer membrane still contains slight contaminations of LPS, but its strong cytokine-inducing ability in mononuclear cells, which is not found in the LPS-specific Limulus amebocyte lysate test, indicates an LPS-independent mechanism of cell activation.

Bacterial Outer Membrane Proteins↗
PubMedDate not supplied

[Bacterial endotoxinemia in children with intestinal dysbacteriosis].

34 children with gastrointestinal diseases of infectious, allergic and mixed etiology were examined. The state of normal microflora in the large intestine as indicated by fecal bacterial charts and the level of secretory immunoglobulin A (sIgA) in the contents of the intestine as indicated by the results of radial immunodiffusion were studied. In addition, the content of endotoxin in the children's plasma was determined with the use of the Limulus (LAL) test. The presence of endotoxin in the plasma of children with intestinal dysbiosis was determined in 71.1% of cases. The frequency of the detection of antigenemia was found to be related to the severity of manifestations of dysbiotic changes in the intestine and to the level of sIgA in fecal supernatants. The inclusion of the probiotic preparation Bifidumbacterin forte containing live bifidobacteria adsorbed on activated charcoal into the complex therapy of digestive tract diseases ensured a decrease in the detection rate of endotoxinemia, which correlated with the tendency towards the normalization of defective intestinal microflora.

Adolescent↗
PubMed2001

Determination of endotoxins in the vital pulp of human carious teeth: association with pulpal pain.

OBJECTIVES: The aims of this investigation were to determine the presence or absence of endotoxins in the pulp of symptomatic and symptom-free human carious teeth, to quantify the amount of endotoxins present, and to associate the presence of endotoxins with the acute pulpal pain. MATERIAL AND METHODS: Pulpal tissue was sampled from 28 single-rooted carious teeth (15 symptomatic, 13 symptom-free) derived from 28 patients. Samples were also taken from the pulp of 5 noncarious control teeth. During sampling an effort was made to collect an equal weight of pulpal tissue in all cases (approximately 8 mg). The extraction of endotoxins was performed with the use of phenol-water. The assay and quantitative determination of endotoxins was performed with the use of a limulus lysate test. The data were analyzed statistically by using the independent t test. RESULTS: Endotoxins were detected in pulpal tissues of all carious teeth in the symptomatic (mean average, 0.15773 ng/mL; SD = 0.045811) and symptom-free group (mean average, 0.10723 ng/mL; SD = 0.010925). In noncarious control teeth, endotoxins were not detected. The presence of endotoxins was significantly higher in the group of symptomatic teeth than in the group of symptom-free teeth (P <.001). CONCLUSIONS: The presence of endotoxins in the pulpal tissue of all the carious teeth indicates that they may play a major role in the pathogenesis of human pulpal diseases. Since a significantly higher level of endotoxins was detected in the pulp of symptomatic carious teeth than in that of symptom-free carious teeth, an association of endotoxins levels with severity of pulpal pain is probable.

Adolescent↗
PubMed1989

Non-septic endotoxemia in cirrhotic patients.

We have found that endotoxemia detected by conventional LCT (limulus colorimetric test) in patients with liver diseases could not be detected by endotoxin-specific LCT at all, and proposed that this beta-glucan like activity (BGLA) should be termed as non-septic endotoxemia, distinguishing it from septic endotoxemia seen in gram-negative sepsis. In this study, we investigated non-septic endotoxemia through the clinical course of 8 cirrhotic patients. Non-septic endotoxemia appeared at the onset of DIC but tended to decline in level in the late terminal stage. This phenomenon cannot be consistent with the "spillover" theory which explains the mechanism of endotoxemia without sepsis in liver disease. We think it is an urgent problem to elucidate the nature of BGLA in liver disease, without recourse to the "spillover" theory.

Adult↗
PubMed1982

Postischemic liver damage in rats: effect of some therapeutic interventions on survival rate.

The aim of this paper is to elucidate the cause of death after 90 min of normothermic partial (2/3) ischemia of the liver and to examine the effects of glucagon, somatostatin, insulin, prednisolone and oral administration of polymyxin B (PB). The animals 24 hr after partial ischemia for 90 min were divided into two groups; namely, animals with normal appearance and those with moribund state. There were no significant differences in the plasma level of S-GOT, S-GPT, amino acids, NH3 or insulin, or in morphometrically estimated volume ratio of necrotic hepatocytes between the two groups of rats. The blood glucose level, however, was significantly decreased (31 +/- 28 mg/100 ml, n = 6) in the moribund rats with a higher incidence of positive Limulus gelation tests as compared with the rats with normal appearance (149 +/- 19, n = 5). The 1-day and 1-week survival rates of the animals were 42/62 (69%) and 32/61 (53%), respectively. A glucagon injection (1.5 mg/kg, after ischemia) was effective to elevate the 1-day survival rate (14/14), but failed to increase the 1-week survival rate (11/14). On the other hand, a somatostatin injection (100 micrograms/kg, after ischemia) or PB treatment (15 mg/kg/day x 5-9, before ischemia) succeeded to increase the 1-week survival rate (20/22 p less than 0.01 and 17/17 p less than 0.01, respectively), although no significant amelioration in transaminase levels or volume ratio of necrosis was demonstrated. It could be seen that a moribund state after partial ischemia was accompanied by severe hypoglycemic shock, and that the injection of somatostatin after ischemia or the annihilation of gram-negative bacteria by means of oral administration of polymyxin B before ischemia prevented the occurrence of the hypoglycemic shock.

Amino Acids↗
PubMed1999

[Determination of TNF-alpha and endotoxemia in patients with chronic liver diseases].

The incidence of endotoxemia in 153 patients with different liver diseases was determined by Limulus Lysaite Test(LLT). Comparative studies were carried out on the levels of tumour necrosis factor-alpha(TNF-alpha) between positive and negative endotoxemia in patients suffering from chronic liver diseases. At the same time, 58 patients of positive endotoxemia were divided into two groups--bifidobiogen group (38 cases) and control group(20 cases). The patients in bifidobiogen group were treated mainly with daily administration of bifidobiogen for 1 month besides general liver--protection therapy and the bifidobiogen was not given to the control group. The results showed that: 1. Incidence of endotoxemia in 153 patients suffering from various types of liver diseases was 65.36%; the highest rate was found in the patients with severe hepatitis; 2. The TNF-alpha level was obviously increased in the patients of positive endotoxemia; 3. The total efficacy in bifidobiogen group was significantly higher than that in control group. The data suggest that chronic liver diseases are generally complicated with endotoxemia, and TNF-alpha plays an important role in the pathogenesis of endotoxemia. The serum endotoxin level may be reduced after the treatment with bifidobiogen.

Adult↗
PubMed1982

Lack of correlation between endotoxaemia and renal hypoperfusion in cirrhotics without overt renal failure.

Renal involvement in patients with liver cirrhosis is characterized by renal vasoconstriction, the aetiology of which remains obscure. Endotoxaemia, frequently found in patients with liver cirrhosis and renal failure, has been emphasized as a pathogenic factor. In fifty-seven patients with liver cirrhosis without overt renal failure endotoxin plasma level (Limulus Lysate test), mean renal blood flow (MRBF) (133Xe washout technique), and effective renal plasma flow (ERPF) (p-aminohippurate clearance) were determined. MRBF was decreased in nineteen out of twenty-seven patients, averaging 1.88 +/- 0.51 ml g-1 min-1 (in fourteen controls 3.17 +/- 0.51 ml g-1 ml-1). ERPF was decreased in seventeen out of thirty patients, averaging 380 +/- 164 ml/min (in eighteen controls 624 +/- 127 ml/min). Systemic endotoxaemia was found in sixteen out of fifty-seven patients, levels ranging from 0.62 to 200 ng/ml. No significant difference in renal blood flow values was found between patients with and without endotoxaemia (MRBF = 1.78 +/- 0.51 and 1.93 +/- 0.52 ml g-1 min-1 respectively; ERPF = 429 +/- 119 and 365 +/- 175 ml/min respectively). No significant difference in the frequency of endotoxaemia was found between patients with impaired and unimpaired renal blood flow. Moreover no relation was found between endotoxin plasma levels and MRBF and ERPF respectively. In conclusion in patients with cirrhosis without overt renal failure renal vasoconstriction does not seem to be related to endotoxaemia.

Adult↗
PubMed1992

Toxicity and immunogenicity of Neisseria meningitidis lipopolysaccharide incorporated into liposomes.

To obtain nontoxic and highly immunogenic lipopolysaccharide (LPS) for immunization, we incorporated Neisseria meningitidis LPS into liposomes. Native LPS and its salts were incorporated by the method of dehydration-rehydration of vesicles or prolonged cosonication. The most complete incorporation of LPS into liposomes and a decrease in toxicity were achieved by the method of dehydration-rehydration of vesicles. Three forms of LPS (H+ form, Mg2+ salt, and triethanolamine salt) showed different solubilities in water, the acidic form of LPS, with the most pronounced hydrophobic properties, being capable of practically complete association with liposomal membranes. An evaluation of the activity of liposomal LPS in vitro (by the Limulus amoebocyte test) and in vivo (by monitoring the pyrogenic reaction in rabbits) revealed a decrease in endotoxin activity of up to 1,000-fold. In addition, the pyrogenic activity of liposomal LPS was comparable to that of a meningococcal polysaccharide vaccine. Liposomes had a pronounced adjuvant effect on the immune response to LPS. Thus, the level of anti-LPS plaque-forming cells in the spleens of mice immunized with liposomal LPS was 1 order of magnitude higher and could be observed for a longer time (until day 21, i.e., the term of observation) than in mice immunized with free LPS. The same regularity was revealed in a study done with an enzyme-linked immunosorbent assay. This study also established that antibodies induced by immunization belonged to the immunoglobulin M and G classes, which are capable of prolonged circulation. Moreover, liposomal LPS induced a pronounced immune response in CBA/N mice (defective in B lymphocytes of the LyB-5+ subpopulation). The latter results indicate that the immunogenic action of liposomal LPS occurs at an early age.

Animals↗
PubMed1989

Overall study of the in vitro plasma clotting system in an invertebrate, Liocarcinus puber (Crustacea Decapoda): considerations on the structure of the Crustacea plasma fibrinogen in relation to evolution.

An overall study of the in vitro plasma coagulation system in the crab Liocarcinus puber has been carried out using various analytical methods, namely thromboelastography, spectrophotometrical examination, and a new one based on changes of the mechanical impedance of the developing clot. From the results reported here the clotting pattern in this species appears surprisingly complex for an invertebrate and unexpectedly closer to that of the vertebrates. Indirect evidences suggest that the fibrinogen polypeptide chains in this species and very likely in the other crustacean, are very different from those of the vertebrates. This would imply that crustacean and vertebrate fibrinogen would have diverged from one another in a far remote past, far beyond the individualization of the vertebrate alpha chain, that is, over 1.5 million years ago.

Animals↗
PubMed1977

Comparison of the limulus amebocyte lysate (LAL) test with the rabbit test: false positives and false negatives.

Although the test for pyrogens on rabbits, described by the European Pharmacopoeia, is efficient for the detection of clinically important concentrations of pyrogens, it has some practical disadvantages; for instance, it blocks the further use of a rabbit for three weeks once this has been used to test a solution which proved to contain pyrogens. The LAL test for bacterial endotoxin lipopolysaccharides was explored as an alternative method for the detection of pyrogens in infusion fluids and medicinal preparations. False negatives in the LAL test were obtained on some corticosteroid preparations and on some infusion fluids from a batch which had caused febrile reactions in the clinic.

Animals↗