Bacterial endotoxin is considered as one of the critical risk factors in medical devices, especially implanted devices that directly or indirectly contact with blood circulating system. In that case, endotoxin limits for implanted medical devices is important in determine the safety of medical devices. According to GB/T 14233.2-2005, the requirements of endotoxin index for intrathoracic medical devices is 2.15 EU per device. However, the definition of "intrathoracic medical devices" is vague. Specifically, "for cardiovascular system application" instead of "intrathoracic application" is more reasonable. With the deeper understanding of the risk of endotoxin in medical devices and considering the internationally accepted standards, the limits of endotoxin in medical devices for cardiovascular system application is acceptable at 20 EU per device.
Endotoxins

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 observe the binding power of polymyxin B (PMB) and its simulation peptide to lipopolysaccharide (LPS) and lipoid A.

METHODSLPS and lipoid A were separately coated on biosensor. 5 microl of PMB (0.01 microg/L) 5 microl of its simulating peptide 1 (PMBSP1 0.01 microg/L) and 5 microl of its simulating peptide 2 (PMBSP2, 0.01 microg/L) were respectively added into the hydrophobic sample pool. The combining power of PMB and its simulating peptides PMBSP1 and PMBSP2 to LPS and lipoid A was compared. RESULTS (1) PMBSP1 almost did not bind LPS and lipoid A, while PMB and PMBSP2 possessed high affinity with LPS and lipoid A. (2) The peak value (98.41 +/- 7.31) rad/s of PMBSP2 binding LPS was much higher than that (83.58 +/- 5.42) rad/s of PMB in binding LPS (P < 0.05). While the peak value of PMB in binding lipoid A was similar to that of PMBSP2. (3) The peak value of PMB binding LPS was significantly lower than that of PMB in binding lipoid A (P < 0.05). But there was no difference between the peak value of PMBSP2 in binding LPS and that of PMBSP2 in binding lipoid A. (4) PMBSP2 could bind to LPS and lipoid A in a shorter time to reach peak levels.

CONCLUSIONCompared with PMB, the PMBSP2 could bind to LPS and lipoid A in a shorter time. In addition, PMBSP2 exhibited similar affinity to LPS and lipoid A. This indicated that PMBSP might possess better anti-LPS activity due to its lack of space steric hindrance when PMBSP binding the lipoid A of LPS.

Cell Wall ; chemistry ; Endotoxins ; Gram-Negative Bacteria ; Lipopolysaccharides ; chemistry ; toxicity ; Peptides ; chemistry ; pharmacology ; Polymyxin B ; chemistry ; pharmacology

Ovarian cancer (OC), the deadliest gynecological cancer, results in poor overall survival, urgently requiring a novel therapeutic approach. As cumulative exposures to endotoxins decreased OC risk epidemiologically, we evaluated if LPS, a Toll-like receptor 4 agonist known as active component of endotoxins, could increase survival in the murine peritoneal dissemination model of SKOV-3 OC cells. LPS significantly increased the mean survival time of more than 116 days compared with 63 days in the control. Furthermore, no tumor burden was present in three mice among eight LPS-treated mice. SKOV-3 cells were not responsive to LPS and showed unaltered chemokine signature. Rather than direct effects to OC cells, LPS was found to increase proinflammatory chemokines and cytokines, such as CXCL1, CXCL8, TNF, and IL-1B, in innate immune system. Taken together, LPS is likely to potentiate the cytotoxic-related innate immunogenicity via proinflammatory chemokines and cytokines, which attenuates the peritoneal dissemination of OC.
Animals ; Chemokines ; Cytokines ; Endotoxins ; Immune System ; Immunity, Innate ; Lipopolysaccharides ; Mice ; Ovarian Neoplasms ; Survival Rate ; Toll-Like Receptor 4 ; Tumor Burden

OBJECTIVETo explore the application of biosensor technology in the determination of endotoxin-neutralizing materials.

METHODSAfter mixing polymyxin B (PMB) with endotoxin in certain concentration, the neutralizing ratio of PMB to endotoxin was assessed by biosensor technique and limulus amebocyte lysate test respectively. The results from the two methods were compared.

RESULTSThe neutralizing ratio of PMB to endotoxin as assessed by biosensor technology was 0.35 microg to 1 ng, while that by dynamic turbidimetric and chromogenic limulus amebocyte lysate (LAL) technique was 0.5 mg to 1 ng and 1 mg to 1 ng, respectively. The results obtained by biotechnology were similar to that by biosensor technique.

CONCLUSIONBiosensor technology was an accurate, convenient and rapid method for the determination of potency of endotoxin-neutralizing materials.

Bacterial Proteins ; analysis ; Biosensing Techniques ; methods ; Endotoxins ; analysis ; Lipid A ; analysis ; Polymyxin B ; analysis ; Reproducibility of Results ; Sensitivity and Specificity

Baker's asthma is the most prevalent occupational asthma, and IgE-mediated response is known as a major pathogenesis. However, recent studies have suggested the involvement of innate immune response because wheat flour contains bacterial endotoxins or lipopolysaccharides. To further understand a role of innate immune response in the development of work-related respiratory symptoms (WRS) in bakery workers, we investigated mannose-binding lectin (MBL), one of the initiating components of the complement cascade in a single cohort of bakery workers. A total of 373 bakery workers completed a questionnaire regarding WRS. The bakery workers were divided into 2 groups according to previous history of allergic rhinitis (AR)/bronchial asthma (BA): those with history of AR/BA (group I) and those without (group II). We measured serum MBL levels by using enzyme-linked immunosorbant assay and genotyped 4 single nucleotide polymorphisms of the MBL2 gene (226G>A in exon 1, -554G>C, -431A>C, and -225G>C in the promoter) by using TaqMan assays. Fifty-nine subjects (15.5%) were previously diagnosed with AR/BA, and 64 subjects (16.8%) complained of WRS. No significant differences were found in serum MBL levels between groups I and II. However, in group II subjects, but not in group I subjects, the serum MBL levels were significantly higher in bakery workers with WRS than in those without. In addition, the serum MBL levels were significantly different according to genetic polymorphisms of the MBL2 gene and its haplotypes. In conclusion, serum MBL, affected by genetic polymorphisms, may be associated with WRS in bakery workers with no previous history of AR/BA.
Asthma ; Asthma, Occupational ; Cohort Studies ; Complement System Proteins ; Endotoxins ; Exons ; Flour ; Haplotypes ; Immunity, Innate ; Lipopolysaccharides ; Mannose-Binding Lectin ; Polymorphism, Genetic ; Polymorphism, Single Nucleotide ; Rhinitis, Allergic ; Triticum

OBJECTIVETo explore the mechanism of endotoxin tolerance (ET) through observing the expression of interleukin 1 receptor associated kinase-4 (IRAK-4) during endotoxin tolerance development in Kupffer cells (KCs).

METHODSIsolated KCs of Balb/c mouse were divided into two groups: the non-endotoxin tolerance (NET) group and the endotoxin tolerance (ET) group, which were pretreated with 10 ng/ml lipopolysaccharide (LPS) for 24 h. Then, the two groups were treated with 100 ng/ml LPS. The expressions of IRAK-4 gene and protein level were determined by RT-PCR and Western blot. The activities of NF-kappaB of KCs and the TNFalpha level were estimated by ELISA at 0 h, 1 h, 3 h, 6 h and 12 h after LPS stimulation.

RESULTSThe ultimate level of IRAK-4, the activities of NF-kappaB and the TNFalpha level were evidently lower in the ET group than those in the NET group (t = 12.4, 17.4 and 138.9 respectively, P<0.01).

CONCLUSIONSPretreatment with LPS on KCs could induce endotoxin tolerance of KCs and inhibition of IRAK-4 expression may be one of the reasons for its development.

Animals ; Cells, Cultured ; Endotoxins ; immunology ; Immune Tolerance ; Interleukin-1 Receptor-Associated Kinases ; biosynthesis ; genetics ; Kupffer Cells ; cytology ; immunology ; metabolism ; Lipopolysaccharides ; immunology ; Male ; Mice ; Mice, Inbred BALB C

Obesity and metabolic syndrome is a worldwide pandemic and associated with high cardiovascular risk. Metabolic endotoxemia (ME) is thought to be an underlying molecular mechanism. It triggers toll-like receptor 4-mediated inflammatory adipokines and causes a chronic low grade inflammatory status, which results in cardiovascular risk increase. Exercise is the best nonpharmacological treatment to improve prognosis. In this study, we examined the circulating endotoxin level in Korean obese women and investigated effects of exercise on it. Women over body mass index (BMI) 25 kg/m2 participated in a resistance training exercise, Curves. At baseline and after 12 weeks exercise, tests including blood samples were taken. In Korean obese women, the fasting endotoxin was 1.45 ± 0.11 EU/mL. Ingestion of a high calorie meal led to a peak level after 2 hours (postprandial 2 hours [PP2]) and a significant rise over the 4 hours (postprandial 4 hours [PP4]) in it (1.78 ± 0.15 and 1.75 ± 0.14 EU/mL for PP2 and PP4, P < 0.05 vs. fasting). After exercise, BMI and hip circumference were reduced significantly. The total cholesterol (TC) at fasting, PP2 and PP4 were decreased significantly. All levels of circulating endotoxin at fasting, PP2 and PP4 showed reduction. But, the peak change was only significant (baseline vs. 12 weeks for PP2; 1.78 ± 0.15 vs. 1.48 ± 0.06 EU/mL, P < 0.05). We report the circulating endotoxin level in Korean obese women for the first time. Also, we establish that energy intake leads to endotoxemia and exercise suppresses the peak endotoxemia after meal. It suggests an impact for a better prognosis in obese women who follow regular exercise.
Adipokines ; Body Mass Index ; Cholesterol ; Eating ; Endotoxemia* ; Endotoxins ; Energy Intake ; Fasting ; Female ; Gastrointestinal Microbiome ; Hip ; Humans ; Lipopolysaccharides ; Meals ; Obesity* ; Pandemics ; Prognosis ; Resistance Training ; Toll-Like Receptors

OBJECTIVETo investigate the enhancing effect of Chinese medicine-Xuebijing injection on lipopolysaccharide (LPS) -induced apoptosis of CD4+ CD25+ regulatory T cells (Tregs) and polarization of helper T cells (Th).

METHODSCD4+ CD25+ Tregs collected from rat spleen in vitro by immunomagnetic beads assay were divided into the control group, anti-CD3/CD28 group, anti-CD3/CD28 + LPS group, anti-CD3/CD28 + "Xuebijing injection" group and anti-CD3/CD28 + LPS + "Xuebijing injection" group. Tregs apoptosis rate and expression of winged helix transcription factor (Foxp3) in Tregs were detected by flow cytometry on 3rd post culture day. CD4+ CD25- T cells were co-cultured with CD4+ CD25- Tregs (1:1) for 68 hours with canavalin A stimulation. Interferon gamma (gamma-IFN), interleukin (IL)-4 and IL-17 in supernatants, which respectively was secreted by Th1, Th2 and Th17, were measured by ELISA.

RESULTSTregs apoptosis rate of anti-CD3/CD28 + LPS + "Xuebijing injection" group (45.1 +/- 2.7%) was significantly higher than that of anti-CD3/CD28 + LPS group (29.4 +/- 1.6%, P < 0.01). Meanwhile, Foxp3 expressions in Tregs in above 2 groups were 95 +/- 9 and 140 +/- 18 respectively, showing statistically significant difference between them (P < 0.01). Gamma-IFN levels secreted in anti-CD3/CD28 + LPS + "Xuebijing injection" group were significantly higher than those in anti-CD3/CD28 + LPS group (P < 0.01), while IL-4 levels had an opposite tendency compared with gamma-IFN (P < 0.05), resulting in a marked increase in the ra- tio of gamma-IFN/IL-4 in anti-CD3/CD28 + LPS + "Xuebijing injection" group (P < 0.01). In anti-CD3/ CD28 + "Xuebijing injection" group, IL-17 secretion levels were significantly decreased compared with anti-CD3/CD28 group (P < 0.05).

CONCLUSIONSActivation of CD4+ CD25+ Tregs induced by LPS may mediate Th1 shift to Th2 response. "Xuebijing injection" can effectively regulate immune function of T cells, increase the LPS-induced apoptosis of CD4+ CD25+ Tregs as well as enhance the polarization of Th2 to Th1, thereby abating the suppressive state of cell-mediated immunity.

Animals ; Apoptosis ; Drugs, Chinese Herbal ; pharmacology ; Endotoxins ; Lipopolysaccharides ; Male ; Rats ; Rats, Wistar ; T-Lymphocytes, Helper-Inducer ; drug effects ; immunology ; T-Lymphocytes, Regulatory ; drug effects ; immunology

OBJECTIVETo observe the effect of mastoparan-1 (MP-1) on lipopolysaccharide (LPS)-induced acute hepatic injury in mice and probe into its possible mechanism.

METHODSOne hundred and four BALB/c mice were randomly divided into healthy control group (n = 8, without treatment, HC), LPS group (n = 48, with injection of LPS 5 mg/kg via tail vein), and MP-1 group (n = 48, with injection of LPS 5 mg/kg and MP-1 3 mg/kg via tail vein). Mice in LPS group and MP-1 group were sacrificed at 2nd, 6th, 12th, 24th, 48th and 72nd post injection hour (PIH), 8 mice at each time point in each group. Blood samples were collected for determination of plasma levels of LPS by kinetic turbidimetric limulus test, TNF-alpha and IL-6 by ELISA, serum levels of ALT and AST by automatic biochemistry analyzer respectively. Hepatic tissue samples were collected for determination of TLR4, TNF-alpha and IL-6 mRNA by real-time fluorescent quantitation reverse transcription polymerase chain reaction, along with the observation of pathological changes in hepatic tissue at each time point. Above-mentioned examinations were also performed in HC group.

RESULTSCompared with those of HC group, plasma levels of LPS and TNF-alpha in LPS group significantly increased at 2nd PIH (18,320.50 +/- 2782.50 EU/mL and 988 +/- 130 ng/L, respectively), then decreased gradually to 1.80 +/- 0.80 EU/mL and 150 +/- 44 ng/L at 72nd PIH, which was close to those of HC group. The values of IL-6, ALT and AST peaked at 12th PIH, which declined to the levels close to those of HC group at 72nd PIH. Meanwhile, the expressions of TLR4, TNF-alpha and IL-6 mRNA in liver were remarkably up-regulated after injection, and the pathological changes in hepatic tissue pronounced significantly at 12th, 24th and 48th PIH. Compared with those of LPS group, the levels of LPS, cytokines, ALT and AST decreased in MP-1 group in different degrees after injection (P < 0.05 or P < 0.01), genes expression (P < 0.05 or P < 0.01) and pathological changes was respectively suppressed and alleviated in hepatic tissue.

CONCLUSIONSMP-1 can alleviate LPS-induced acute hepatic injury in mice, which may be associated with its neutralization of LPS and attenuation of synthesis and release of inflammatory mediators.

Animals ; Chemical and Drug Induced Liver Injury ; pathology ; Endotoxins ; adverse effects ; Inflammation ; Lipopolysaccharides ; adverse effects ; Liver ; drug effects ; pathology ; Mice ; Mice, Inbred BALB C ; Peptides ; pharmacology ; Wasp Venoms ; pharmacology