OBJECTIVETo detect content of bacterial endotoxin in Yuxingcao and Qingkailing injections by specific and nonspecific tachypleus amebocyte lysate technique for in order to investigate the feasibility of specific tachypleus amebocyte lysate technique for detecting bacterial endotoxin in traditional Chinese drug injections.

METHODDifferent batches of Yuxingcao and Qingkailing injections were detected by specific and nonspecific tachypleus amebocyte lysate kits.

RESULTYuxingcao injection could be detected by specific and nonspecific tachypleus amebocyte lysate technique, Whereas Qingkailing injection could be detected only by specific tachypleus amebocyte lysate.

CONCLUSIONUsing specific tachypleus amebocyte lysate as a substitute for nonspecific tachypleus amebocyte lysate is an effective method for detecting content of bacterial endotoxin in Qingkailing injection.

Animals ; Drug Contamination ; Drugs, Chinese Herbal ; analysis ; Endotoxins ; analysis ; Horseshoe Crabs ; Limulus Test ; methods

OBJECTIVETo investigate the role of REMP2 derived from limulus anti-lipopolysaccharide factor in neutralizing endotoxin in vitro and its antibacterial activity.

METHODS(1) REMP2 and PMB in the concentrations of 100.00, 10.00, 1.00, 0.10, 0.01 micromol/L were respectively mixed with LPS (lEU/mL), with PMB as positive control. The LPS concentrations in different specimens were determined by routine method, and the neutralizing percentage was respectively calculated. (2) After adding isotonic saline (NS), the final concentrations of REMP2 and PMB were 10, 20, 40, 80 micromol/L, and the concentration of LPS was 100 microg/L. The murine monocytic macrophages were stimulated with LPS, then cultured with REMP2 and PMB, with NS in culture as negative control. The content of tumor necrosis factor (TNF)-alpha was determined by ELISA kit. (3) The morphologic changes of Escherichia coli. was observed under electron microscope at 10, 20 and 40 minutes after addition of REMP2 to Escherichia coli suspension (with terminal concentration of REMP2 at 40 micromol/L).

RESULTSThere were no significant difference in endotoxin-neutralizing percentages between PMB and REMP2 in concentrations of 0.10, 10.00, 100.00 micromol/L (P > 0.05). The contents of TNF-alpha were 1175 +/- 162, 859 +/- 122, 645 +/- 142, 489 +/- 102 ng/L, respectively,after treatment of 10, 20, 40, 80 micromol/L REMP2, which were obviously lower than that of NS (3463 +/- 218 ng/L, P < 0.01). Under transmission electron microscope, the outer and interior membranes of Escherichia coli were obscure and rough, bacterial bodies were swollen with vacuoles in cytoplasm after treatment with REMP2.

CONCLUSIONREMP2 has ability of neutralizing endotoxin and also antibacterial activity.

Animals ; Anti-Bacterial Agents ; pharmacology ; Antimicrobial Cationic Peptides ; Arthropod Proteins ; Cells, Cultured ; Escherichia coli ; drug effects ; metabolism ; Invertebrate Hormones ; pharmacology ; Limulus Test ; Lipopolysaccharides ; antagonists & inhibitors ; Macrophages ; metabolism ; Mice ; Peptide Fragments ; pharmacology ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo investigate the mechanism of polymyxin B (PMB) antagonizing the biological activity of lipopolysaccharide (LPS).

METHODSThe affinity of PMB for LPS and lipid A was assayed by biosensor, and the neutralization of PMB for LPS (2 ng/ml) was detected by kinetic turbidimetric limulus test. The releases of TNF-alpha and IL-6 in murine peritoneal macrophages a (PMphi) after exposure to LPS (100 ng/ml) were detected, and the expression levels of TLR4, TNF-alpha and IL-6 mRNA in PMphi induced by LPS (100 ng/ml) were measured by RT-PCR.

RESULTSPMB had high-affinity to LPS and lipid A with dissociation equilibrium constants of 18.9 nmol/L and 11.1 nmol/L, respectively, and neutralized LPS in a dose-dependent manner. Furthermore, PMB could markedly inhibit the expressions of TLR4, TNF-alpha and IL-6 mRNA and the release of cycokines in LPS-stimulated murine peritoneal macrophages.

CONCLUSIONSPMB neutralizes LPS and inhibites the expression and release of cycokines in macrophages, in which the affinity of PMB for lipid A plays an important role.

Animals ; Cytokines ; analysis ; Limulus Test ; Lipid A ; antagonists & inhibitors ; Lipopolysaccharides ; antagonists & inhibitors ; Macrophages ; chemistry ; Mice ; Polymyxin B ; pharmacology

OBJECTIVETo study the anti-endotoxin of different concentration baicalin.

METHODS6.250 microg/mL, 3.125 microLg/mL, 1.562 microg/mL and 0.781 microg/mL baicalin solutions were mixed with I EU/mL endotoxin, respectively. The mixtures were put into water of (37+/-1) degrees C for 15 min, 30 min and 60 min. The degrading effects were determined by using limulus amebocyte lysate test (LAL test).

RESULTS1) The degrading effect of 6.250 microg/mL, 3.125 microg/mL and 1.562 microg/mL baicalin solution on I EU/mL endotoxin was degraded completely in 15 min, 30 min and 60 min, respectively. 2)The degrading effect of 3.125 microg/mL, 1.562 microg/mL and 0.781 microg/mL baicalin solution on 1 EU/mL endotoxin after these mixtures had been incubated for 15 min. Endotoxin values were (0.155 5 +/- 0.002 8) EU/mL, (0.212 1+/-0.004 9) EU/mL, (0.355 9+/-0.013 9) EU/mL, respectively. These differences among them were statistically significant (P<0.01). 3) The degrading effect of 1.562 microg/mL and 0.781 microg/mL baicalin solution on 1 EU/mL endotoxin after these mixtures had been incubated for 30 min. Endotoxin values were (0.1640+/-0.0025) EU/mL and (0.2094+/-0.004 4) EU/mL, respectively. These differences between them were statistically significant (P<0.01).

CONCLUSIONThe action of anti-endotoxin of baicalin is dose-dependent and time-dependent. The results show that baicalin has the stronger anti-endotoxin effect.

Endotoxins ; Flavonoids ; Limulus Test

OBJECTIVETo investigate the lipopolysaccharide (LPS) antagonizing biological activity of densefruit pattany root-bark extract (DPR-2) in vitro.

METHODSThe effect of DPR-2 in neutralizing LPS (0.1 microg/L) was detected by kinetic turbidimetric limulus test. The effect of different concentrations of DPR-2 (0,8.0,16.0,32.0,64.0 mg/L) on binding of FITC-conjugated LPS (FITC-LPS,100.0 microg/L) to murine RAW264.7 cells was analyzed with laser scanning confocal microscopy. The expression of TNF-alpha and IL-6 mRNA in RAW264.7 cells after exposure to LPS (100.0 microg/L) were determined by real-time RT-PCR.

RESULTSDPR-2 could neutralize LPS (P < 0.05 or P < 0.01), and inhibit the binding of FITC-LPS to RAW264.7 cells in a dose-dependent manner when the concentration of DPR-2 was above 16.0 mg/L. Furthermore, DPR-2 could markedly inhibit the expression of TNF-alpha and IL-6 mRNA in LPS-stimulated murine RAW264.7 cells.

CONCLUSIONDPR-2 exhibit an anti-LPS effect in vitro, which may be related to its capacity to neutralize LPS and inhibit binding of LPS for its receptors.

Animals ; Cell Line ; Drugs, Chinese Herbal ; pharmacology ; Endotoxins ; antagonists & inhibitors ; In Vitro Techniques ; Limulus Test ; Lipopolysaccharides ; antagonists & inhibitors ; Mice ; Monocytes ; drug effects ; metabolism ; Plant Extracts ; pharmacology

OBJECTIVETo determine bacterial endotoxin in the replacement solution of on-line hemodiafiltration (on-line HDF) using kinetic turbidimetric limulus test.

METHODS AND RESULTSValidation test was performed with the replacement solution of on-line HDF in which quantified standard endotoxin was added. The recovery rates of endotoxin from the replacement solution and its dilutions at 1/5, 1/10, and 1/20 were 58.17%, 106.7%, 99.00% and 98.79%, respectively, suggesting that the optimal dilution was at 1/10. Standard endotoxin was added into the replacement solution of on-line HDF of 3 batches (040408, 040511,040527), and the recovery rates in their dilution at 1/10 were 76.32%, 99.00% and 96.24%, respectively. The standard endotoxin in the working curve was 1.00, 0.125, and 0.0156 Eu/ml (endotoxin unit/ml), and the dilution at 1/10 of the replacement solution is effective to eliminate the interference in limulus test.

CONCLUSIONKinetic turbidimetric limulus test provide a means to detect endotoxin in the replacement solution of on-line HDF.

Endotoxins ; analysis ; Hemodiafiltration ; methods ; Hemodialysis Solutions ; analysis ; Humans ; Kinetics ; Limulus Test ; Nephelometry and Turbidimetry ; methods

Based on the biological activity of endotoxin, we propose a possible new method for detecting endotoxin using a pH- indication system of macrophage culture media. After RAW 264.7 macrophage cells were treated with lipopolysaccharide (LPS), the addition of fluorescein to the LPS-treated media reproductively reduced its absorption and emission spectra (it was a dose-dependent reduction). The advantages of this LPS- detection method were compared with the Limulus Amebocyte Lysate (LAL) test by using purified bacterial LPS (Salmonella minnessota, Escherichia coli, and Pseudomonas aeruginosa). Additionally, the absorption and fluorescence intensity of fluorescein, following treatment of RAW 264.7 cells with a high concentration of Staphylococcus aureus (Gram-positive, lysed bacteria), could not generally be detected by the LAL test, but they were found to be reduced, in a dose-response relationship, with this new system. The macrophage culture system-method might be a good supplement to the LAL assay for detection of LPS, Gram-negative and Gram-positive bacteria.
Animals ; Biological Assay/*methods/standards ; Cells, Cultured ; Comparative Study ; *Contrast Media ; Culture Media/chemistry ; Endotoxins/*analysis ; *Fluorescein ; Hydrogen-Ion Concentration ; Limulus Test ; Lipopolysaccharides/analysis ; Macrophages/*chemistry ; Mice ; Research Support, Non-U.S. Gov't

OBJECTIVETo compare the endotoxin levels between acute and chronic periapical periodontitis with different clinical symptoms.

METHODS10 cases of acute periapical priodontitis(Group 1), 10 cases of chronic periapical periodontitis (group 2, the diameter of apical radiolucency area was less than 2 mm), 10 cases of chronic periapical periodontitis with sinus(group 3, the diameter of apical radiolucency area was greater than 2 mm), 10 cases of chronic periapical periodontitis without sinus (group 4, the diameter of apical radiolucency area was greater than 2 mm), were included in the study. Chromogenic substrate method of limulus amebocyte lysate(LAL) test was used to measure the endotoxin level.

RESULTSEndotoxin concentrations in group 2 were significantly lower than those in group 1, group 3 and group 4(P < 0.01).

CONCLUSIONEndotoxin plays a very important role in the initiation and development of periapical periodotitis and is closely associated with clinical symptoms and apical radiolucency degree.

Acute Disease ; Bacteria ; Chronic Disease ; Endotoxins ; analysis ; Humans ; Limulus Test ; Periapical Periodontitis ; metabolism ; microbiology ; Root Canal Irrigants

To establish stable methods for detecting plasma endotoxin level and endotoxin inactivation capacity in a normal population and general surgical patients and evaluate their perioperative changes. 50 healthy people and 50 patients receiving gastrointestinal operation were enrolled, their plasma endotoxin levels and plasma endotoxin inactivation capacity were assayed. Our results showed that plasma endotoxin levels were 0.044 +/- 0.009 EU/ml in the normal population and 0.044 +/- 0.023 EU/ml in the preoperative patients. Endotoxin level peaked 3 h after the operation (0.223 +/- 0.041 EU/ml), and then decreased rapidly on the first day after the operation (0.134 +/- 0.164 EU/ml). Endotoxin inactivation capacity also had the same time course as endotoxin level. Systemic inflammatory response syndrome and infection induced another elevation in the time course. It is concluded that establishing the endotoxin standard curve by using pyrogenic free water is better than by using plasma. Plasma endotoxin inactivation capacity can be used as an indirect indicator of postoperative immune depression. Plasma endotoxin level and endotoxin inactivation capacity peaked shortly after operation, indicating surgical stress is closely related with the changes.
Adult ; Aged ; Colorectal Neoplasms ; blood ; surgery ; Endotoxins ; blood ; Female ; Humans ; Limulus Test ; methods ; Male ; Middle Aged ; Postoperative Period ; Reference Values ; Stomach Neoplasms ; blood ; surgery ; Stress, Physiological ; blood ; Systemic Inflammatory Response Syndrome ; blood

The aim of this study was to evaluate the morphological and biological properties of a locally produced "Bovine Bone Sponge" for use in dentistry. Bovine bone sponge was prepared from local calf bone. Endotoxin level and surface properties were investigated. The pore size and water uptake ability were measured and results were compared with the commercial haemostatic agent. The material was tested for its haemostatic property and its inhibition of alveolar bone resorption in a sheep model following dental extraction. Results revealed a significant difference in haemostatic effect, and a shorter bleeding time and a lower rate of alveolar bone resorption in bovine bone sponge compare to a commercial haemostatic agent.
Alveolar Bone Loss/prevention & control ; *Biocompatible Materials ; *Bone and Bones ; Endotoxins/analysis ; Hemorrhage/*therapy ; *Hemostatics ; Limulus Test ; Microscopy, Electron, Scanning ; Sheep ; *Surgical Sponges ; *Tooth Extraction