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PubMed1983

Role of bacterial endotoxins of intestinal origin in rat heat stress mortality.

Using unanesthetized rats, the effect on heat stress mortality of endotoxin tolerance or zymosan treatment was determined. In addition, the incidence of invasion by gram-negative bacteria and their endotoxins was studied to evaluate the role of gut-derived bacterial endotoxins after heat stress. Endotoxin tolerance resulted in heat stress resistance. The estimated mean total thermal area, which induced an LD50 in endotoxin-tolerant rats (61.85 degrees C . min) was significantly greater (P less than 0.001) than that for non-tolerant rats (44.03 degrees C . min). Rats were significantly (P less than 0.005) more sensitive to endotoxin after zymosan treatment, but this treatment did not alter the heat stress mortality rate. The Limulus amoebocyte lysate test indicated that endotoxemia did not occur as a result of heat stress. Though a significantly increased incidence of high gram-negative bacterial count in the duodenum was noted, extraintestinal invasion was not found. It was concluded that resistance to heat stress may not be due to protection from gut-derived bacterial endotoxins, but resistance may possibly be associated with the ability of endotoxin tolerance to protect from shock syndromes. Thus bacterial endotoxins of intestinal origin did not appear to have a significant role in rat heat stress mortality.

Animals↗
PubMed1999

Endotoxin and cytokine release in strangulation obstruction and in partial occlusion of the mesenteric artery in pigs.

BACKGROUND: The study was performed to determine if endotoxin or the cytokines interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor (TNF) are liberated from strangulated or partially ischemic small bowel. METHODS: Strangulation obstruction was induced by elevating pressure in a gasket placed around a loop of ileum until venous pressure reached 50 mm Hg. Low arterial flow in a loop of ileum was produced by arterial clamping reducing blood flow by 70%. A proximal bowel loop was used for control. Arterial blood flow was measured by transit time flowmetry. Blood samples were collected before and after 30, 90 and 180 min of strangulation or clamping. Plasma levels of endotoxin and cytokines (TNF, IL-1 and IL-6) were measured by limulus amebocyte lysate test and bioassays, respectively. RESULTS: Strangulation obstruction caused more extensive mucosal damage than arterial clamping. Strangulation was followed by markedly increased venous concentration and release of IL-6 in the strangulated loop. Partial arterial occlusion did not cause increased release of IL-6. Strangulation or partial clamping did not influence the concentration of endotoxin, IL-1 or TNF in intestinal venous blood. CONCLUSIONS: Strangulation obstruction causes increased release of IL-6 to intestinal venous blood. IL-6 levels did not increase after 70% reduction of arterial blood flow. The early IL-6 increase was not detected in systemic blood. Strangulation did not cause early changes in plasma levels of endotoxin, TNF or IL-1.

Animals↗
PubMed1996

Determination of endotoxin-neutralizing capacity of plasma in postsurgical patients.

In 92 patients who underwent abdominal and goiter surgery endotoxin and endotoxin-neutralizing capacity (ENC) were determined in plasma preoperatively and daily postoperatively. Endotoxin plasma levels started to increase on day 1 in patients who were laparotomized. Correspondingly ENC was reduced during the 1st postoperative week. Even on the 1st postoperative day determination of ENC made the differentiation between patients possible who showed an uneventful course and patients who developed pneumonia, pulmonary failure, or mental disorders. Furthermore the need for diuretics in order to maintain sufficient renal function was associated with lower ENC during days 1-3. Endotoxin plasma levels were significantly elevated in patients who developed pneumonia during days 1-4, whereas the occurrence of pulmonary failure was only correlated with elevated endotoxin levels on day 3. Endotoxemia was pronounced in patients needing diuretics on days 1, 4, and 5. In patients who underwent goiter surgery ENC changed significantly as found for endotoxin plasma values during the postoperative course. Determination of endotoxin and especially ENC with the use of the limulus amebocyte lysate test turned out to be a reliable method correlating with impending complications at least in postsurgical patients.

Adult↗
PubMed2002

House dust endotoxin and allergic sensitization in children.

A higher exposure to endotoxin was hypothesized to contribute to lower prevalence of allergic sensitization and hay fever in children growing up on a farm. We studied the association between house dust endotoxin and allergic sensitization. We randomly selected 740 children, aged between 5 and 10 years, from a group of children who participated in two cross-sectional surveys performed in Saxony-Anhalt, Germany, from 1992 to 1993 and from 1995 to 1996, such that 50% of the children were atopic or had a diagnosis of asthma. From 1996 to 1998, we collected living-room floor dust in the homes of 454 of these children (61%). The content of endotoxin in house dust was quantified using a chromogenic kinetic limulus amoebocyte lysate test and was related with health outcomes measured in the preceding cross-sectional surveys. Multiple logistic regression analyses adjusted for place of residence, sex, age, parental education, parental atopy, and pet ownership showed a negative association between exposure to endotoxin and sensitization to one or more allergens (aOR [95% CI] 0.95 [0.83; 1.10]) and two or more allergens (aOR [95% CI] 0.80 [0.67; 0.97]) using 0.35 kU/L as the cutoff value for sensitization. The protective effect was strengthened with increasing degree of sensitization. In conclusion, exposure to higher levels of house dust endotoxin is associated with lower prevalence of allergic sensitization in children.

Air Pollution, Indoor↗
PubMed2003

Removal of endotoxin by reverse phase HPLC abolishes anti-endothelial cell activity of bacterially expressed plasminogen kringle 5.

The success of recombinant protein expression/purification in Escherichia coli depends on a high-fidelity system rendering purified proteins free of confounding contaminants such as endotoxin. Here we report on the expression and purification of a cryptic plasminogen-derived domain, kringle 5, which was previously reported to specifically inhibit endothelial cell growth and, therefore, angiogenesis. Using a histidine (HIS)-tag expression and Ni(+)-NTA agarose purification system identical to previous reports, we found that our purified recombinant kringle 5 did inhibit endothelial cell growth, but this activity could not be eradicated by heat denaturing or proteolysis of kringle 5 with various proteases. This led us to suspect the presence of a contaminant in the purified samples. Quantitative endotoxin testing using a limulus amoebocyte lysate assay revealed that all samples purified by Ni(+)-NTA agarose alone harbored high concentrations of endotoxin that could not be removed by additional purification on anion exchange chromatography. Finally, when kringle 5 was rendered endotoxin-free by purification on reverse phase high-performance liquid chromatography (HPLC), there was a complete loss of endothelial cell growth inhibitory activity. These results strongly suggest that endotoxin-free recombinant kringle 5 may not possess anti-angiogenic activity and demonstrates that, especially in angiogenesis type assays, endotoxin contamination can lead to a misinterpretation of results.

Animals↗
PubMed2000

Evaluation of heparin immobilized chitosan-PEG microbeads for charcoal encapsulation and endotoxin removal.

A technique is described to encapsulate activated charcoal for hemoperfusion to be used in an artificial liver support. Activated charcoal was encapsulated within chitosan-PEG matrix and subsequently surface modified with PGE1 or heparin (hep-AC-PEGCB) via the glutaraldehyde functionalities. This novel matrix was used as the supports for perfusion of endotoxin, under a flow rate of 30 ml/mt. Endotoxin adsorption was quantitatively measured by the method of Limulus Amebocyte lysate test. It seems, the hep-AC-PEGCB may be a good adsorbent system for the removal of toxic endotoxin, and the system may be useful for detoxification of blood. The hep-AC-PEGCB matrix had improved biocompatibility as demonstrated from their hemolytic potential and charcoal release. However, further studies are needed to determine their behaviour under clinical conditions.

Alprostadil↗
PubMed1989

Endotoxin release from neisseria meningitidis. Relationship between key bacterial characteristics and meningococcal disease.

A method was established in order to measure total and filtrable (CF) endotoxin in cultures of Neisseria meningitidis strains. The Limulus lysate (LAL) test gave results which paralleled those of the standard rabbit pyrogen test and of gas chromatography (GLC), concerning detection of different CF endotoxin levels. Meningococci varied in their ability to produce CF endotoxin. Cultures of similar bacterial densities, whether with a high (E+) or a low (E-) release of endotoxin in CF, had both high yields of endotoxin in sonicated culture suspensions determined by the LAL test. GLC demonstrated only small differences in total LPS contents between E+ and E- strains. This suggests that strains with similar cell wall endotoxin contents may vary in CF endotoxin. Electron microscopy revealed that E+ strains presented a high number of free, outer cell wall fragments (blebs, tubuli, membranes and aggregates of such structures) in surroundings. Few such free, small structures were found around E- strains. The amount of CF endotoxin of E+ strains was in part a function of the number of colony forming units (CFU/ml), and generally followed the growth curve. Because of its moment of appearance, and also based on electron microscopy findings, CF endotoxin appeared mainly to be released from living bacteria. The CF level of endotoxin was low or not detected at all in cultures of E- strains although their cultures reached higher mean CFU-levels than the niveaus required for the detection of CF endotoxin in the E+ strains. The E+ property was strain dependent. Meningococci isolated from CSF or blood had a significantly higher proportion of E+ strains (88.2%) and a higher CF endotoxin titre (greater than or equal to 10(3); 34.5%), than isolates from carriers (32.3% and 10.8%, respectively) (p less than 0.001 and p less than 0.001, respectively). A high mean CFU/ml in cultures seemed to be more often associated with isolates from patients than from carriers, more often with the presence than abscence of capsular polysaccharide (p less than 0.05), and more often with the presence than absence of the E+ property (p = 0.002). E+ strains were mostly serogroupable (i.e. encapsulated), regardless of source of the isolate (99% case and 80% carrier isolates). In contrast, serogroupable bacteria were not necessarily E+ when isolated from carriers (54.8%). The serogroup most apt to cause disease tended to have the highest proportion of E+ strains and the highest level of CF endotoxin.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2006

[Study on pyrogen in natural and cultivated edible mushrooms].

We examined endotoxin and pyrogen contents in several kinds of natural and cultivated edible mushrooms, as well as some cultivated vegetables. According to the Japanese Pharmacopoeia, 14th Ed., two types of endotoxin (gel-clot Limulus amebocyte lysate) test and the pyrogen test were performed using natural edible mushrooms collected in Aichi Prefecture and cultivated mushrooms and vegetables purchased at a market. The endotoxin contents of natural mushrooms were apparently higher than those of cultivated mushrooms or vegetables. The endotoxin contents in the cultivated mushrooms were slightly higher than those in the vegetables. Similar results were obtained in the pyrogen test.

Agaricales↗
PubMedDate not supplied

[Possible contamination of preparation of transfer factor with bacterial endotoxins].

In order to detect the possible contamination with bacterial endotoxins of some preparations of Transfer Factor, Limulus amebocyte lysate test was performed. The results obtained (TF preparations positive with L.T. for endotoxin assay) call sur detection to evaluate very carefully the immunologic responses in vitro ad in vivo, with these biological products contaminate can induce. In fact, bacterial endotoxins with their multiple potent effect on biologic system, represent a contant possible surce of variability in experimental biology and pathology.

Drug Contamination↗
PubMed1999

[Orienting endotoxin measurement in the atmosphere].

Airborne endotoxins are supposed to influence the respiratory health of animal and man in animal housings as well as at certain work places in agriculture. Little is known about the usual concentrations of endotoxin in the outdoor atmosphere. Therefore in a field study 30 air samples were taken, 22 samples in a rural region, and 8 in a more residential and industrial area. The samples were taken by impingement and filtration. The analysis was carried out by means of the chromogen-kinetic limulus amoebocyte lysate test (LAL-Test). The median concentration of all samples was 0.36 ng/m3. The highest median concentration of 0.49 ng/m3 was found in summer with a maximum value of 1.80 ng/m3 indicating large variations. In the other seasons the median concentrations (ng/m3) were distinctly lower (spring: 0.30, n = 11; autumn: 0.26, n = 5; winter: 0.19, n = 3). No significant differences were observed between farming and residential areas at this low concentration level. The concentrations of endotoxin found in this study were far below all threshold limits which are presently discussed for work places.

Air Pollutants↗
PubMed2001

Endotoxin, endotoxin-neutralizing-capacity, sCD14, sICAM-1, and cytokines in patients with various degrees of alcoholic liver disease.

BACKGROUND: Chronic alcohol ingestion leads to endotoxemia which is believed to play an important role in the pathogenesis of alcoholic liver disease (ALD). The purpose of this study was to determine if chronic ethanol consumption, in addition to affecting plasma endotoxin and cytokines, also affects the endotoxin-neutralizing capacity (ENC), sCD14, and sICAM-1, in patients with ALD. A second aim was to identify correlations between these latter parameters, endotoxin, and cytokines, especially IL-10. METHODS: Hospitalized patients with various degrees of ALD (n = 59), and 20 healthy volunteers were studied. Plasma endotoxin and ENC were determined using our kinetic Limulus amebocyte lysate test. Cytokines, sCD14, and sICAM-1 were measured by enzyme-linked immunosorbent assay. RESULTS: Patients with ALD exhibited a mild endotoxemia (p < 0.01) and a marked decrease in ENC (p < 0.0002). TNF-alpha (p < 0.05), IL-6 (p < 0.0001), sICAM (p < 0.005), and sCD14 (p < 0.0005) were significantly elevated in all patients with ALD, and IL-10 (p < 0.05) in patients with cirrhotic ALD. With the exception of IL-10, the cytokines correlated with each other and with sICAM-1. No correlations occurred between endotoxin, ENC, and sCD14, and between these and the cytokines and sICAM-1. Elevated levels of endotoxin correlate with acute excessive alcohol ingestion. No gender differences were observed. CONCLUSIONS: Acute alcohol intoxication rather than severe ALD results in significant endotoxemia. The limited capacity of plasma to neutralize endotoxin in liver injury seems to be an important factor in ALD which may be responsible for the release of endotoxin-induced mediators, such as cytokines, as well as s-ICAM-1, that are relevant in the pathogenesis of ALD.

Alcoholic Intoxication↗
PubMed1999

[The relationship between tissue tumor necrosis factor-alpha(TNF-alpha) mRNA expression and gut-derived endotoxemia in rats after thermal injury].

OBJECTIVE: To investigate the dynamic changes in tumor necrosis factor-alpha(TNF-alpha) mRNA expression and the relationship between TNF-alpha mRNA and gut-derived endotoxemia in rats after thermal injury. METHODS: 56 male Wistar rats were subjected to a 35% total body surface area full-thickness thermal injury and randomly divided into 3 groups: normal controls, 35% III* injury group, and polymyxin B treatment group. Plasma endotoxin was measured by limulus amebocyte lysate test, and tissue TNF-alpha mRNA expression was determined by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: It was found that a significant elevation of endotoxin concentration in both portal and systemic circulation was observed at 2 hours after thermal injury, peaking at 8 hours (P < 0.05-0.01) and lowering at 24 hours. Meanwhile, TNF-alpha mRNA expression significantly increased in liver, lung, intestine, and kidney at 2 hours, peaking at 8 hours, and a high level was maintained till 24 hours. However, treatment with a low-dose polymyxin B could lower both portal and systemic endotoxin levels, and markedly inhibit TNF-alpha mRNA expression in various organs. In addition, a positive correlation between portal endotoxin concentration and pulmonary, intestinal and renal TNF-alpha mRNA expression was noted (r = 0.365-0.484, all P < 0.05). CONCLUSION: This study suggests that major burns can lead to a marked elevation of tissue TNF-alpha mRNA expression in vital organs, which may be associated with gut-derived endotoxemia caused by acute thermal injury.

Animals↗
PubMed2000

MALP-2, a Mycoplasma lipopeptide with classical endotoxic properties: end of an era of LPS monopoly?

Although some activities of LPS are shared by other bacterial components, for half a century LPS has been regarded as unique in displaying many pathophysiological activities. Here we report on a synthetic lipopeptide, MALP-2 from Mycoplasma fermentans, which expresses potent endotoxin-like activity and whose lethal toxicity is comparable to that of LPS. With the exception of the Limulus lysate gelation test, in which MALP-2 was approximately 1000-fold less active than LPS, the synthetic lipopeptide induced all activities tested for, and in most cases to an extent comparable to that of LPS. Unlike LPS, the biological activities of MALP-2 were expressed both in LPS-responder and in LPS-non-responder mice (BALB/c/l, C57BL10/ScCr), indicating that MALP-2 signaling, unlike that of LPS, is not transduced via the Toll-like receptor (Tlr) 4 protein.MALP-2 expressed no toxicity in normal or sensitized Tlr2 knockout (Tlr2(-/-)) mice indicating that its toxic activity is induced via Tlr2 signaling. The phenomenology of the lethal shock induced by MALP-2 in normal or sensitized mice, i.e. the kinetics of its development and symptoms of illness exhibited by the treated animals, was very reminiscent of the lethal shock induced by LPS.

Animals↗
PubMed1998

Protective effect of bactericidal/permeability-increasing protein in mice with E. coli sepsis.

OBJECTIVE: To investigate the effect of bactericidal/permeability-increasing protein(BPI) on the outcome of sepsis in mice and its possible mechanism. METHODS: Sepsis was induced by injection of 2x10(6) colony-formed unit E. coli J5 via the tail vein. BPI of 5 mg/kg or equal volume of normal saline(NS) were injected intravenously at the same time. Endotoxin and TNFalpha levels in serum were assayed using a chromogenic limulus amebocyte lysate test and ELISA respectively. RESULTS: Seventy-two hour survival rate of septic mice was significantly higher in the BPI group (15/18) than in the NS group(8/18, P<0.01). Serum endotoxin levels in the BPI group (1.3+/-0.3 and 0.7+/-0.4 &mgr;g/L) were significantly lower than those in the NS group (3.9+/-0.8 and 2.5+/-0.9 &mgr; g/L, P<0.01) 0.5 and 1 hour following injection of bacteria respectively. The peak levels of serum tumor necrosis factor-alpha(TNFalpha)in the BPI group (1.9+/-0.6 &mgr;g/L) were also markedly lower than those in the NS group (3.8+/-0.8 &mgr;g/L, P<0.01) 1.5 hours following bacterial injection. But there was no significant difference in blood bacterial count between the BPI and NS groups 0.5, 1.5 and 3.0 hours after injection of bacteria. CONCLUSIONS: BPI has a marked protective effect on E. coli sepsis, which might be related to its action against bacterial endotoxin and its inhibition of TNFalpha production in sepsis.

Journal Article↗
PubMed2002

Enteral application of an immunoglobulin-enriched colostrum milk preparation for reducing endotoxin translocation and acute phase response in patients undergoing coronary bypass surgery--a randomized placebo-controlled pilot trial.

OBJECTIVE: To evaluate the influence of enteral application of an immunoglobulin enriched bovine milk preparation on endotoxin plasma levels, endotoxin neutralizing capacity of plasma (ENC) and the acute phase response (IL-6, CRP) during and after cardiac surgery, in a pilot study. DESIGN, PATIENTS AND METHODS: 60 patients who underwent coronary bypass operations, were evenly enrolled in a placebo-controlled randomized study. The patients were treated by enteral application of either 42 g of a bovine colostrum milk preparation per day or placebo, for 2 days preoperatively. Endotoxin and ENC were sequentially determined intra- and postoperatively by a chromogenic modification of the limulus amebocyte lysate test. Interleukin-6, CRP, transferrin, alpha-2-macroglobulin, albumin, apo-A, apo-B, IgG, IgA, IgM were determined by ELISA and nephelometrically. The clinical course was followed up by daily evaluation of the Apache-II-score. MAIN RESULTS: Demographic data were comparable in both groups. No differences of the Apache-II-score (verum group: 6.5 +/- 1.9 vs. controls: 6.8 +/- 1.8 on admission) were observed. Endotoxin and ENC levels were elevated at the end of the operation and seemed to have a trigger function for the acute phase response. However, there was no reduction (calculated as the area under the curve) in patients receiving the colostrum milk preparation throughout the observation period. Plasma levels of endotoxin binding proteins did not differ. Plasma IL-6 concentrations increased to maximal median values of 655 pg/ml in the verum and 786 pg/ml in the control group, respectively 2 and 6 h after surgery. In the colostrum group, there was a tendency to reduced IL-6 levels throughout the observation period. CRP-levels of all patients peaked 48 h after the operation but were lower (p = 0.034) in the verum group. CONCLUSION: This study revealed that endotoxemia occurs early in an elective non-intestinal surgical intervention, and is followed by a subsequent increase in mediators of the acute phase reaction. The prophylactic enteral application of a bovine milk preparation for two days in cardiac patients did reduce postoperative CRP-plasma levels but, contrary to a former double-blind study in abdominal surgery, failed to curtail perioperative endotoxemia. One reason could be the amount of colostrum preparation administered was too small.

APACHE↗
PubMed1999

[Extraction and characterization of the lipopolysaccharide of Bartonella quintana]

Bartonella quintana has been reported as the cause of trench fever, persistent endocarditis, bacteriaemia and has been isolated with an increasing incidence in clinical specimens from AIDS patients. One of the main pathogenic factors of gram-negative bacteria, including B. quintana, is the lipopolysaccharide (LPS). However, very little information is available on the features of Bartonella LPS. The aim of the present study was to extract, purify and characterise B. quintana LPS. The effect of the LPS under scrutiny was also evaluated on TNFa release by means of the "in vitro" human whole blood model of sepsis. The Oklahoma strain of B. quintana was grown on sheep blood agar, at 37 C, in a moist atmosphere containing 5% carbon dioxide. Cells were harvested and washed in sterile and apyrogenic saline solution and LPS extracted following the procedure of Westphal e Jann (1965), modified by Minnick (1994). The LPS of B. quintana showed the migration pattern of a deep rough chemotype, and the chromogenic limulus amoebocyte lysate test (LAL test) revealed strong reactivity at low concentrations (6.2 pg/ml). Samples of human whole blood stimulated by 1000 ng/ml of B. quintana LPS released 1707 378 pg/ml of TNFa.

Journal Article↗
PubMedDate not supplied

Endotoxin in patients with terminal renal failure undergoing dialysis with re-processing dialyser.

AIM: to determine the level of endotoxin in the blood of patients with renal failure prior to and following hemodialysis using re-processing dialyser to know possibility of pyrogenic reactions in hemodialysis patients. METHODS: this study subjects consisted of 10 patients with terminal renal failure undergoing regular hemodialysis. The collected samples were then sent in frozen condition for endotoxin examination in Japan. The normal level of endotoxin in the blood was < 9.8 pg/ml based on standard E.Coli E.0111 endotoxin quantitatively measured using Limulus Amoebocyte lysate test (the endospecy test). Statistical analysis was performed using paired student test. RESULTS: Ten patients with terminal renal failure who were undergoing hemodialysis were obtained, consisting of 1 female and 9 males. The mean age was 55.5 years (SD 6.74), the mean hemoglobin level 7.26 g/dl (SD 2.19), mean white blood cell (WBC) count 8660/mm(3) (SD 3064.2), and mean albumin level 3.59 g/l (SD 247). The etiologies of renal failure were as follows: glomerulonephritis (GN) 30%, Diabetic nephropathy (DN) 20%, hypertension (HT) 10%, interstitial nephritis (IN) 10%, obstruction/infection (01) 10%, unknown (U) 10%. The mean duration of hemodialysis was 97.9 month (SD 54.86). The mean endotoxin level prior to hemodialysis (ET pre-hemodialysis) was 5.4 pg/dl (SD 8). CONCLUSION: we conclude that terminal renal patients who undergoing re-processing hemodialysis did not have endotoxemia both prior to and following hemodialysis unless if they associated with infection, or other complications.

Aged↗
PubMed1990

[Immunoglobulin therapy of postoperative sepsis].

35 patients with septic postoperative complications entered a prospectively randomized study. The clinical state of these patients was daily determined using the sepsis score described by Elebute and Stoner. Endotoxin and antithrombin III were measured in the plasma using the limulus-amoebocyte-lysat test for endotoxin determination. The septic patients were treated with an immunoglobulin preparation (Pentaglobin) administered by the intravenous route. This preparation is enriched in IgM and IgA. Due to the immunoglobulin therapy the endotoxin titres decreased; simultaneously a reduction of mortality and shortening of time of hospitalization and of mechanical ventilation were observed.

Antithrombin III↗