Limulus Amebocyte Lysate Assay Research Articles

Membrane filtration enhanced by magnetic field for reducing endotoxin from dialysis water

AimThis study investigates the impact of magnetic field-assisted filtration on endotoxin reduction in dialysis water, focusing on the efficacy of ultrafiltration and nanofiltration membranes. MethodsThe starting material was synthetic water with a known amount of endotoxin (0.48 EU/ml). The experiment was conducted in a small-lab-scale unit, likely to test and optimize the purification method before large-scale application. Limulus amebocyte lysate (LAL) assay is a standard test used to detect and quantify endotoxin levels in liquids. ResultsThe study found that all treatment strategies effectively reduced endotoxin levels, with the best treatment being a third hybrid treatment involving magnetic field, UF, and NF exposure. This kind of treatment reduced the endotoxin from 0.169 ± 0.09 EU/ml to 0.06 ± 0.06 EU/ml at an exposure time of 20 min for a magnetic field. ConclusionThe integration of magnetically treated water in dialysis treatment plants holds promise for enhancing the quality of dialysis fluid and aligning with international standards. By employing physical methods like magnetic treatment, facilities can address concerns surrounding water purity in dialysis, which is crucial for patient safety and treatment effectiveness.

A Study on the Application of Recombinant Factor C (rFC) Assay Using Biopharmaceuticals.

Gram-negative bacterial endotoxins can cause pathophysiological effects such as high fever when introduced into the bloodstream. Therefore, endotoxin testing is necessary when producing injectable pharmaceuticals. The pharmaceutical industry has widely used Limulus amebocyte lysate (LAL) to certify product quality. However, ethical concerns have been raised and the increasing scarcity of Limulus polyphemus necessitates the development of novel testing techniques. Recombinant factor C (rFC) was developed using genetic engineering techniques. The aim of this study was to investigate the validity of rFC testing and compare it with the LAL method. The specificity, linearity, accuracy, precision, and robustness of the rFC assay were evaluated. After validation, the rFC assay was found to be suitable for endotoxin detection. We compared the accuracy of the rFC and LAL assays using reference standard endotoxin. The rFC assay was as accurate as the LAL assay. We also compared the two assays using biopharmaceuticals. Greater interference occurred in some samples when the rFC assay was used than when the LAL assay was used. However, the rFC assay overcame the interference when the samples were diluted. Overall, we suggest that rFC can be applied to test biopharmaceuticals.

Heparin enables the reliable detection of endotoxin in human serum samples using the Limulus amebocyte lysate assay

The determination of lipopolysaccharide (endotoxin) in serum or plasma samples using Limulus amebocyte lysate (LAL)-based assays is currently not sufficiently reliable in clinical diagnostics due to numerous interfering factors that strongly reduce the recovery of LPS in clinical samples. The specific plasma components responsible for the endotoxin neutralizing capacity of human blood remain to be identified. There are indications that certain endotoxin-neutralizing proteins or peptides, which are part of the host defense peptides/proteins of the innate immune system may be responsible for this effect. Based on our finding that several antimicrobial peptides can be neutralized by the polyanion heparin, we developed a heparin-containing diluent for serum and plasma samples, which enables reliable quantification of LPS measurement in clinical samples using the LAL assay. In a preclinical study involving 40 donors, this improved protocol yielded an over eightfold increase in LPS recovery in serum samples, as compared to the standard protocol. This modified protocol of sample pretreatment could make LPS measurement a valuable tool in medical diagnostics.

Rational design of antibodies and development of a novel method for (1-3)-β-D glucan detection as an alternative to Limulus amebocyte lysate assay.

With advances in medicine, increasing medical interventions have increased the risk of invasive fungal disease development. (1-3)-β-D glucan (BDG) is a common fungal biomarker in serological tests. However, the scarcity of Limulus resources for BDG detection poses a challenge. This study addresses the need for an alternative to Limulus amebocyte lysate by using BDG mutant antibody for chemiluminescence detection. The wild-type BDG antibody was obtained by immunizing rabbits. An optimal V52HI/N34L Y mutant antibody, which has increased 3.7-fold of the testing efficiency compared to the wild-type antibody, was first achieved by mutating "hot-spot" residues that contribute to strong non-covalent bonds, as determined by alanine scanning and molecular dynamics simulation. The mutant was then applied to develop the magnetic particle chemiluminescence method. 574 clinical samples were tested using the developed method, with a cutoff value of 95 pg/mL set by Limulus amebocyte lysate. The receiver operating characteristic curve demonstrated an area under the curve of 0.905 (95% CI: 0.880-0.929). Chemiluminescence detected an antigen concentration of 89.98 pg/mL, exhibiting a sensitivity of 83.33% and specificity of 89.76%. In conclusion, the results showed a good agreement with Limulus amebocyte lysate and demonstrated the feasibility of using BDG mutant antibodies for invasive fungal disease diagnosis. The new method based on chemiluminescence for detecting BDG could shorten the sample-to-result time to approximately 30 min, rescue Limulus from being endangered and is resource efficient in terms of equipment and the non-use of a skilled technician.

Current Considerations and Practical Solutions for Overcoming Nanoparticle Interference with LAL Assays and Minimizing Endotoxin Contamination.

Monitoring endotoxin contamination in drugs and medical devices is required to avoid pyrogenic responses and septic shock in patients receiving these products. Endotoxin contamination of engineered nanomaterials and nanotechnology-based medical products represents a significant translational hurdle. Nanoparticles often interfere with an in vitro limulus amebocyte lysate (LAL) assay commonly used in the pharmaceutical industry for the detection and quantification of endotoxin. Such interference challenges the preclinical development of nanotechnology-formulated drugs and medical devices containing engineered nanomaterials. Protocols for the analysis of nanoparticles using LAL assays have been reported before. Here, we discuss considerations for selecting an LAL format and describe a few experimental approaches for overcoming nanoparticle interference with the LAL assays to obtain more accurate estimations of endotoxin contamination in nanotechnology-based products. The discussed approaches do not solve all types of nanoparticle interference with the LAL assays but could be used as a starting point to address the problem. This chapter also describes approaches to prevent endotoxin contamination in nanotechnology-formulated products.

Effect of Surgery on Postoperative Levels of the Gut Homeostasis-Regulating Enzyme Intestinal Alkaline Phosphatase.

Intestinal homeostasis is a crucial factor for complication-free short- and long-term postoperative recovery. The brush border enzyme intestinal alkaline phosphatase (IAP) is an important regulator of gut barrier function and intestinal homeostasis and prevents endotoxemia by detoxifying lipopolysaccharides (LPSs). As IAP is predominantly secreted by enterocytes in the duodenum, we hypothesized that pancreaticoduodenectomy (PD) leads to a significantly stronger decrease in IAP than other major abdominal surgery. Pre- and postoperative blood, stool, and intestinal samples were collected from patients undergoing PD, as well as other major surgical procedures without duodenectomy. The samples were analyzed using enzyme histochemistry, the para -nitrophenyl phosphate method for IAP, and the limulus amebocyte lysate assay for LPS. Overall, 88 patients were prospectively enrolled in the study. Fecal IAP activity negatively correlated with serum LPS (r = -0.3603, p = 0.0006). PD led to a significant decline in IAP compared to preoperative baseline levels (p < 0.0001). The decline in IAP correlated with the length of proximal small intestinal resection (r = 0.4271, p = 0.0034). Compared to controls, PD was associated with a much more pronounced reduction in IAP-also after adjusting for surgical trauma (operative time, blood loss; r = 0.4598, p = 0.0086). Simultaneously, PD triggered a clearly more prominent increase in serum LPS compared to controls (p = 0.0001). Increased postoperative LPS was associated with an elongated hospitalization (r = 0.7534, p = 0.0062) and more prominent in pancreatic cancer (p = 0.0009). Based upon the functional roles for IAP, supplementation with exogenous IAP might be a new treatment option to improve short- and long-term outcome after PD.

Immunostimulatory activity and safety evaluation of Bordetella bronchiseptica-derived lipopolysaccharide, a new vaccine adjuvant candidate

Adjuvants are used to elicit strong immune responses for vaccines that show poor immunogenicity. Previously, we demonstrated that a sonicated bacterin of Bordetella bronchiseptica can be used as a safe adjuvant that enhances the antigen-presenting capability of dendritic cells (DCs). In this study, we purified the lipopolysaccharide (LPS) of B. bronchiseptica (Bb-LPS) and investigated its immunogenic effects on DCs compared to those of Escherichia coli O26:B6 (O26)-derived LPS (O26-LPS), a positive control. Bb-LPS was purified using an LPS extraction kit. Limulus amebocyte lysate assay was performed to determine the optimal concentration of Bb-LPS and O26-LPS for treatment. Bb-LPS increased the metabolic activity of DCs at a concentration of 0 to 250 EU/mL, similar to that of O26-LPS. Bb-LPS significantly increased the expression level of CD40 and CD54, related to the immune responses of DCs. Bb-LPS enhanced the antigen-presenting capability of DCs and significantly increased the interferon-gamma/interleukin-4 ratio of CD4+ T cells co-cultured with DCs to 0.95 (p < 0.05). Moreover, Bb-LPS increased the production of pro-inflammatory cytokines in a safer manner than that obtained by O26-LPS. In vivo safety tests revealed that Bb-LPS was less toxic than O26-LPS in mice. This study demonstrated that Bb-LPS showed unique immune characteristics and immunogenic effects on the antigen-presenting capability of DCs, which differed from those of O26-LPS. This study provides valuable information for basic and clinical research for developing safe vaccine adjuvants.

Endotoxin contamination of nanoparticle formulations: A concern in vaccine adjuvant mechanistic studies.

The increasing awareness of endotoxin contamination has raised important questions during the study of the mechanism of action of the vaccine adjuvants. The endotoxins or lipopolysaccharides (LPS) can contaminate vaccine formulations contributing to result misinterpretations of the in vitro and in vivo studies. In this short communication, we considered the suitability of the Limulus amebocyte lysate (LAL) assay to quantify chitosan (Chit) nanoparticle (NP) endotoxin contamination to use them in a comparative in vitro immunotoxicology study using both LPS-free (LF) and non-LF Chit NPs. It was shown that chit NPs had a masking effect on endotoxin levels, hampering a reliable conclusion about the effect of their contamination. Neither non-LF nor LF Chit NPs induced the production of ROS in RAW 264.7 cells or IL-6 and TNF-α in PBMCs. The lack of effect of non-LF NPs was not expected and likely due to the NPs masking effect, more evident for higher deacetylation degree Chit. Overall, to prevent questionable results, nanomaterials should be produced under endotoxin-free conditions.

Ophthalmic viscoelastics commonly used in cataract surgery: A microbiota investigation.

To survey commonly used, sterile ophthalmic viscoelastic materials used during routine cataract surgery for the presence of bacterial DNA and/or viable bacteria and endotoxin quantification. Samples from three different ophthalmic viscoelastic manufacturers and three different production lots per manufacturer were collected for 16 S ribosomal ribonucleic acid (rRNA) sequencing and conventional aerobic and capnophilic bacterial culture. Other samples of viscoelastic material from the same three manufacturers were collected for endotoxin quantification using a commercially available Limulus amebocyte lysate (LAL) assay. Statistical analysis was performed using Sigma Plot 14.0, and R v4.0.2.0. Differences (p ≤ .05) between sample collection sites in total DNA concentration, microbial richness, mean intra-group distances, and endotoxin quantification alongside reagent controls were evaluated. Culture yielded two isolates, identified as Staphylococcus epidermidis and Bacillus megaterium. 16 S rRNA sequencing revealed no differences between brands in richness or overall composition. The most common bacterial DNA detected across all brands was Staphylococcus sp., Cutibacterium sp., Flavobacterium sp., and Lactobacillus sp. A significant difference was found between the median endotoxin concentration between Anvision and Hyvisc® viscoelastic (Anvision: 0.171 EU/mL, Hyvisc®: 0.03 EU/mL; p < .001). No brand-specific differences in bacterial DNA were detected in the viscoelastic materials. Staphylococcus, Cutibacterium, Flavobacterium, and Lactobacillus were the dominant contributors to the bacterial DNA detected. Although Anvision viscoelastic samples contained significantly more endotoxin than Hyvisc® viscoelastic samples, endotoxin concentrations were below the FDA limit of 0.2 EU/mL for both manufacturers. These data further the understanding of inflammatory outcomes following cataract surgery.

Ambient particle composition and toxicity in 31 major cities in China

Current assessment of air quality or control effectiveness is solely based on particulate matter (PM) mass levels, without considering their toxicity differences in terms of health benefits. Here, we collected a total of 465 automobile air conditioning (AC) filters from 31 major Chinese mainland cities to study the composition and toxicity of PM at a national scale. Dithiothreitol (DTT) assay showed that normalized PM toxicity (NIOG) in different Chinese cities varied greatly from the highest 4.99 × 10−3 for Changsha to the lowest 7.72 × 10−4 for Yinchuan. NIOG values were observed to have significant correlations with annual PM10 concentration (r = -0.416, p = 0.020) and some PM components (total fungi, SO42− and calcium element). The concentrations of different elements and water-soluble ions in PM also varied by several orders of magnitude for 31 cities in China. Endotoxin concentrations in PM analyzed using limulus amebocyte lysate (LAL) assay ranged from 2.88 EU/mg PM (Hangzhou) to 62.82 EU/mg PM (Shijiazhuang) among 31 Chinese cities. Besides, real-time qPCR revealed 10∼100-fold differences in total bacterial and fungal levels among 31 Chinese cities. The concentrations of chemical (water soluble ions and trace elements) and biological (fungi, bacteria and endotoxin) components in PM were found to be significantly correlated with some meteorological factors and gaseous pollutants such as SO2. Our results have demonstrated that PM toxicity from 31 major cities varied greatly up to 6.5 times difference; and components such as fungi and SO42− in PM could play important roles in the observed PM toxicity. The city-specific air pollution control strategy that integrates toxicity factors should be enacted in order to maximize health and economic co-benefits. This work also provides a comprehensive view on the overall PM pollution situation in China.

Fruits of Hippophaë rhamnoides in human leukocytes and Caco-2 cell monolayer models-A question about their preventive role in lipopolysaccharide leakage and cytokine secretion in endotoxemia.

Preparations from Hippophaë rhamnoides L. (sea buckthorn) have been traditionally used in the treatment of skin and digestive disorders, such as gastritis, gastric and duodenal ulcers, uterine erosions, as well as oral, rectal, and vaginal mucositis, in particular in the Himalayan and Eurasian regions. An influence of an aqueous extract from the fruits of H. rhamnoides (HR) on leakage of lipopolysaccharide (LPS) from Escherichia coli through gut epithelium developed from the human colorectal adenocarcinoma (Caco-2) monolayer in vitro and glucose transporter 2 (GLUT2) translocation were the principal objectives of the study. Additionally, the effect of HR on the production of pro- and anti-inflammatory cytokines (interleukins: IL-8, IL-1β, IL-10, IL-6; tumor necrosis factor: TNF-α) by the Caco-2 cell line, human neutrophils (PMN), and peripheral blood mononuclear cells (PBMC) was evaluated. The concentration of LPS on the apical and basolateral sides of the Caco-2 monolayer was evaluated with a Limulus Amebocyte Lysate (LAL) assay. GLUT2 translocation was evaluated using an immunostaining assay, whereas secretion of cytokines by cell cultures was established with an enzyme-linked immunosorbent (ELISA) assay. HR (500 μg/ml) significantly inhibited LPS leakage through epithelial monolayer in vitro in comparison with non-treated control. The treatment of Caco-2 cells with HR (50–100 μg/ml) showed GLUT2 expression similar to the non-treated control. HR decreased the secretion of most pro-inflammatory cytokines in all tested models. HR might prevent low-grade chronic inflammation caused by metabolic endotoxemia through the prevention of the absorption of LPS and decrease of chemotactic factors released by immune and epithelial cells, which support its use in metabolic disorders in traditional medicine.

Fusobacterium nucleatum infection-induced neurodegeneration and abnormal gut microbiota composition in Alzheimer's disease-like rats.

ObjectiveTo explore whether Fusobacterium nucleatum could lead to behavioral and pathological changes in Alzheimer’s disease (AD)-like model rat and whether they could affect the gut microbiota.MethodsThe cognitive ability and alveolar bone loss of Sprague-Dawley (SD) rats were tested by Morris water maze and Micro-CT, respectively. HE staining and immunohistochemistry were used to analyze the pathological changes and Aβ1–42 in brains. Western blot was applied to detect the expression of p-Tau 181 in the brain. Limulus amebocyte lysate assay and PCR were performed to determine serum LPS level and whether F. nucleatum accessed the brain, respectively. The gut microbiota was analyzed by the 16S rRNA gene sequence.ResultsOral infection with F. nucleatum could induce increased alveolar bone loss and learning impairment in AD-like rats. Additionally, F. nucleatum exposure increased the Aβ1–42 expression by about one-fourth (P < 0.05), p-Tau181 by about one-third (P < 0.05), and serum LPS (P < 0.05) in AD-like rats. Moreover, F. nucleatum could change the gut microflora composition in AD-like rats, accompanied by a significant increase in the abundance of Streptococcus and Prevotella.ConclusionOral infection with F. nucleatum could contribute to abnormalities in cognitive ability and pathological change in the brain of AD-like rats, which may be related to abnormal gut microbiota composition.

Comparison of the Chemical Characteristics and Toxicity of PM2.5 Collected Using Different Sizes of Cyclones

Cyclone sampling devices have been helpful in assessing the toxic effects of fine particulate matter (PM2.5). The particle collection efficiency of sampling devices is critical. This study investigated the effect of cyclone size on particle size, chemical composition, and particle toxicity. Three cyclones with different inner diameters (12–68 mm) were tested for penetration using an aerodynamic particle sizer, fluorescent polystyrene latex, and a differential mobility analyzer. The elemental and water-soluble ion compositions of the particles collected by different cyclones were compared. An evaluation of the particles’ toxicity was conducted by comparing the results of dithiothreitol (DTT), limulus amebocyte lysate (LAL), and cell exposure assays. The experimental evaluation showed a 50% cut-size of the cyclones between 0.17–0.28, 0.34–0.36, and 0.70 μm for the small, medium, and large cyclones, respectively. To collect PM2.5 and evaluate separation performance in the real environment, the small and large cyclones were selected according to the particle penetration and flow rate. A comparison of chemical composition and enrichment factor values found that the particles in the small cyclone samples contained smaller and more anthropogenic sources than those in the large cyclone samples. The oxidative potential (OP) measured by the DTT assay of the samples collected using the small and large cyclones differed across sampling periods and associated with the transition metals. The viability of human epithelial A549 cells after exposure to the collected particles using the cyclones was different across sampling periods and associated with OP. The endotoxin concentrations measured in the LAL assay were found only in the large cyclone samples; they affected the estimated level of cytokine based on IL(interleukin)-6 release from human leukemia monocytic (THP-1) cells derived macro-phage-like cells. Regardless of the size, the cyclone techniques used in this study to collect aerosol particles would be a powerful tool for a detailed evaluation of particle toxicity.

Effects of bifid triple viable on chronic low-grade inflammation in obese mice induced by high fat diet

To explore the effects and possible mechanism of bifid triple viable(BTV) on chronic low-grade inflammation and insulin resistance in obese mice induced by high fat diet(DIO). A total of 18 male C57 BL/6 J mice aged 6 weeks were randomly divided into two groups according to their body weight after adaptive feeding for 2 weeks. Six mice in one group were fed with normal rodent diet as control group. Twelve mice in the other group were fed with high fat diet(60% kcal% fat)(HF). After 8 weeks, it was confirmed that body weight and fasting blood glucose of the mice fed with high fat diet were significantly higher than those of the control group(P&lt;0.001), and then they were randomly divided into HF group and HF+BTV group according to the body weight and fasting blood glucose. Six mice in the HF group continued to be fed with high fat diet. Six mice in the HF+BTV group were fed with high fat diet and administered bifid triple viable suspension by gavage(24 mg/kg body weight), once a day. Mice in the control group and HF group were given equal volume of water as control treatment. After 4 weeks of treatment, blood was collected from tail vein, and the fasting blood glucose was measured by automatic glucose meter. Then the animals were killed and collected blood, distal ileum, liver and epididymal white adipose tissue. The tight junctions between the epithelial cells of ileum mucosa were observed by transmission electron microscopy. Serum endotoxin(also called lipopolysaccharide, LPS) levels were determined by limulus amebocyte lysate assay. Immunohistochemical staining was used to observe the macrophage marker F4/80 in liver and epididymal white adipose tissue. Serum fasting insulin levels, tumor necrosis factor-α(TNF-α), interlukin-6(IL-6) and monocyte chemotactic protein-1(MCP-1) in liver and/or epididymal white adipose tissue were detected by ELISA. The index of homeostasis model assessment of insulin resistance(HOMA-IR) was calculated by formula. Compared with the mice fed with normal rodents diet, the tight junctions between the epithelial cells of ileum mucosa were loose, the levels of serum endotoxin, live TNF-α and IL-6, epididymal white adipose tissue TNF-α, IL-6, MCP-1 were significantly elevated(P&lt;0.05), the number of macrophages increased in liver and epididymal white adipose tissue, fasting glucose, fasting insulin levels and HOMA-IR were significantly up-regulated(P&lt;0.01) in the HF group. In comparison with the HF group, the structures of tight junctions between the epithelial cells of ileum mucosa were normalized, the levels of serum endotoxin, liver TNF-α, IL-6 and epididymal white adipose tissue TNF-α, IL-6, MCP-1 were obviously decreased(P&lt;0.05), the number of macrophages reduced in liver and epididymal white adipocytes, fasting insulin levels and HOMA-IR were significantly down-regulated(P&lt;0.05), but had no effect on fasting blood glucose levels in HF+BTV group. Bifid triple viable may protect intestinal mucosa barrier, alleviate metabolic endotoxemia, thus improve chronic low-grade inflammation in liver and adipose tissue, and partially restore insulin sensitivity in DIO mice by regulating gut microbiota.

Positively charged polyvinylidene fluoride (PVDF) membrane: A potential alternative for absorbent paper points in endodontics

Aim The purpose of the study was to evaluate the performance of Polyvinylidene Fluoride (PVDF) as an alternative to paper points (PPs) for endodontic treatments. Methodology The paper points and the PVDF membrane were evaluated for endotoxin binding using Limulus Amebocyte Lysate (LAL) assay. New paper points and the PVDF membrane were evaluated for the presence of endotoxins. Results Absorbency and endotoxin removal with the 0.22µm PVDF membrane was significantly greater than any of the paper points tested. There was significantly more endotoxin found in new paper points compared to the PVDF membrane. Conclusion In conclusion, our study showed that the 0.22μm PVDF membrane was significantly more absorbent and removed more endotoxins than PPs.

A modified dentine infection model with fluorescent lipopolysaccharide and lipopolysaccharides sampling technique to compare XP-Endo finisher and passive ultrasonic irrigation.

The lipopolysaccharides-dentine-infection (LPS-dentine-infection) models and sampling techniques frequently used to evaluate LPS disinfection have limitations. In this study, a LPS-dentine-infection model was devised using fluorescent conjugate LPS. Secondly, a sampling technique using cryogenic grinding for intraradicular LPS analysis was evaluated. Thirdly, the effectiveness of the XP-endo Finisher (XP-EF) was compared with passive ultrasonic irrigation (PUI) in removing LPS from root canal system. Sixty-nine mandibular premolars were submitted to dentine pretreatment and inoculated with fluorescent LPS conjugate (Alexa Fluor® 594). Twenty-three teeth were analysed under confocal laser scanning microscopy (CLSM) to validate this modified LPS-dentine-infection model. Forty-six teeth were randomly divided into two experimental groups: XP-EF (n=23) and PUI (n=23). All teeth were instrumented with XP-endo shaper (XPS; FKG Dentaire) and 2.5% NaOCl. The root canals were sampled with paper points before (s1) and after (s2) instrumentation and after supplemental treatment (s3) with XP-EF and PUI. After s3, all roots were cryogenically ground for intraradicular LPS analysis (s4). Limulus amebocyte lysate assay was used for LPS quantification. The Friedman test was used for differences in LPS among four time-points (s1, s2, s3, and s4). Dunn's test was used for pairwise testing of time-points. The significance level was set at 5% (p < .05). Fluorescent LPS conjugate was detected in 100% of the samples under CLSM with a penetration depth of approximately 400 μm into dentine. Chemo-mechanical preparation using XPS files significantly reduced LPS levels (p < .05). Both the XPS and PUI improved the LPS disinfection (p < .05), with no difference between them (p > .05). LPS was recovered from all samples after cryogenic grinding. The residual amount of LPS detected using the cryogenically sampling technique at s4 was approximately three times greater than with the paper-point sampling technique at s3. This study established a modified LPS-dentine-infection model using fluorescent conjugate LPS, and validated a LPS sampling technique for using cryopulverization intraradicular LPS analysis. Moreover, both the XP-EF and PUI further improved LPS disinfection from the root canals, and the innovative XP-EF was as effective as PUI.

A Water Extract from Chlorella sorokiniana Cell Walls Stimulates Growth of Bone Marrow Cells and Splenocytes.

A water extract derived from the isolated cell walls of Chlorella sorokiniana (C. sorokiniana, Chlorella water extract, CWE) was analyzed for the presence of lipopolysaccharide (LPS)-related material via the Limulus amebocyte lysate (LAL) assay and evaluated for its growth stimulation effect on the bone marrow cells and splenocytes in vitro cell cultures. The extract contained low levels of LPS-related material, and a mass spectrum suggested that the extract contained many components, including a low level of a lipid A precursor, a compound known as lipid X, which is known to elicit a positive response in the LAL assay. Treatment with the CWE dose- and time-dependently stimulated the growth of mouse bone marrow cells (BMCs) and splenocytes (SPLs). Treatment with the CWE also increased specific BMC subpopulations, including antigen-presenting cells (CD19+ B cells, 33D1+ dendritic cells and CD68+ macrophages), and CD4+ and CD8+ T cells, but decreased the number of LY6G+ granulocytes. Treatment with the CWE also increased cytokine mRNA associated with T cell activation, including TNFα, IFNγ, and granzyme B in human lymphoblasts. The present study indicates that the cell wall fraction of C. sorokiniana contains an LPS-like material and suggests a candidate source for the bioactivity that stimulates growth of both innate and adaptive immune cells.

Poria cocos polysaccharides rescue pyroptosis-driven gut vascular barrier disruption in order to alleviates non-alcoholic steatohepatitis

Ethnopharmacological relevancePoria cocos polysaccharides (PCP) are abundant in Poria cocos (Schw.) Wolf (Poria). This is a common traditional Chinese medicine used to treat gastrointestinal and liver diseases. Poria cocos dispel dampness and enhance gastrointestinal functions, strongly affecting the treatment of non-alcoholic fatty liver disease. Still, the mechanism is not yet clear. Aim of the studyThe latest research found that protecting the integrity of the intestinal barrier can slow down the progression of non-alcoholic fatty liver disease (NAFLD). Hence, our research ought to explore the protective mechanism of PCP on the intestinal barrier under a high-fat diet and to clarify the relationship between intestinal barrier damage and steatohepatitis. Materials and methodsH&E staining was done to evaluate pathological damage, whereas Nile red and oil red O staining was conducted to evaluate hepatic fat infiltration. Immunofluorescence staining and immunohistochemical staining were used to detect protein expression and locations. Bone marrow-derived macrophages were isolated for in vitro experiments. ONOO- and ROS fluorescent probes and MDA, SOD, and GSH kits assessed the levels of nitrogen and oxidative stress. LPS levels were detected with a Limulus Amebocyte Lysate assay. The Western blot analysis and reverse transcription-quantitative PCR detected the expression of related proteins and genes. The Elisa kit detected the level of the inflammatory factors in the cell supernatant.For the vivo NAFLD experiments, in briefly, mice were randomly chosen to receive either a High-fat diet or control diet for 12 weeks. Drug treatments started after 4 weeks of feeding. Zebrafish larvae were raised separately in fish water or 7 mM thioacetamide as the control or model group for approximately 72 h. In the therapy groups, different concentrations of PCP were added to the culture environment at the same time. ResultsIn zebrafish, we determined the safe concentration of PCP and found that PCP could effectively reduce the pathological damage in the liver and intestines induced by the NAFLD model. In mice, PCP could slow down weight gain, hyperlipidemia, and liver steatosis caused by a high-fat diet. More importantly, PCP could reduce the destruction of the gut-vascular barrier and the translocation of endotoxins caused by a high-fat diet. Further, we found that PCP could inhibit intestinal pyroptosis by regulating PARP-1. Pyroptosis inhibitors, such as MCC950, could effectively protect the intestinal and liver damage induced by a high-fat diet. We also found that pyroptosis mainly occurred in intestinal macrophages. PCP could effectively improve the survival rate of bone marrow-derived macrophages in a high-fat environment and inhibit pyroptosis. ConclusionsThese results indicated that PCP inhibited the pyroptosis of small intestinal macrophages to protect the intestinal barrier integrity under a high-fat diet. This resulted in decreased endotoxin translocation and progression of steatohepatitis.

Significant and rapid reduction of free endotoxin using a dialkylcarbamoyl chloride-coated wound dressing.

Endotoxin causes inflammation and can impair wound healing. Conventional methods that reduce bioburden in wounds by killing microorganisms using antibiotics, topical antimicrobials or antimicrobial dressings may induce endotoxin release from Gram-negative bacteria. Another approach is to reduce bioburden by adsorbing microorganisms, without killing them, using dialkylcarbamoyl chloride (DACC)-coated wound dressings. This study evaluated the endotoxin-binding ability of a DACC-coated wound dressing (Sorbact Compress, Abigo Medical AB, Sweden) in vitro, including its effect on the level of natural endotoxin released from Gram-negative bacteria. Different concentrations of purified Pseudomonas aeruginosa endotoxin and a DACC-coated dressing were incubated at 37°C for various durations. After incubation, the dressing was removed and endotoxin concentration in the solution was quantified using a Limulus amebocyte lysate (LAL) assay. The DACC-coated dressing was also incubated with Pseudomonas aeruginosa cells for one hour at 37°C. After incubation, the dressing and bacterial cells were removed and shed endotoxin remaining in the solution was quantified. Overnight incubation of the DACC-coated wound dressing with various concentrations of purified Pseudomonas aeruginosa endotoxin (96-11000 EU/ml) consistently and significantly reduced levels of free endotoxin by 93-99% (p<0.0001). A significant endotoxin reduction of 39% (p<0.001) was observed after five minutes. The DACC-coated dressing incubated with clinically relevant Pseudomonas aeruginosa cells also reduced shed endotoxin by >99.95% (p<0.0001). In this study, we showed that a DACC-coated wound dressing efficiently and rapidly binds both purified and shed endotoxin from Pseudomonas aeruginosa in vitro. This ability to remove both endotoxin and bacterial cells could promote the wound healing process.

MO587: Chronic Kidney Disease Induces Endotoxin-Related Activation of The Innate Immune System

Abstract BACKGROUND AND AIMS Chronic inflammation is common in patients with chronic kidney disease (CKD) and is associated with poor outcomes. Although the etiology is only partly understood, the gut–kidney axis is considered an important contributor [1]. Lipopolysaccharide (LPS), also known as endotoxin, is a well-characterized pyrogen found in the exterior cell membrane of most of the gram-negative bacteria. It plays an important role in promoting intestinal inflammatory responses. When absorbed through the intestinal epithelium, LPS induces inflammation by activating macrophages and monocytes. Due to short half-life and semi-quantitative assay characteristics of the Limulus Amebocyte Lysate (LAL) assay, direct quantification of LPS is not suitable to quantify activation of the innate immune system. Lipoprotein-binding protein (LBP), a more stable biomarker, is an acute phase protein produced mostly by the hepatocytes and intestinal epithelial cells and is an essential component of an effective innate immune response to LPS. Circulating LBP significantly enhances the sensitivity of CD14 + cells (mostly monocytes and macrophages) to stimulation by LPS. LBP has been shown to facilitate binding of LPS to CD14 receptor. Levels of LBP peak in serum shortly after endotoxemia and remain increased up to 72 h later. An increased plasma LBP indicates gut epithelial barrier dysfunction [2]. Whether LBP is altered by CKD is not known. METHOD We analyzed the effects of CKD on LBP plasma concentrations. We used samples from the Leuven mild-to-moderate CKD cohort (NCT00441623). To study causality, we used animal models of experimentally induced CKD. To exclude model-related bias, two different rat models of CKD, i.e. 5/6th nephrectomy and adenine supplementation, were used. LBP was measured using commercially available ELISA kits (Hycultbiotech, The Netherlands). RESULTS In a cohort of 460 patients with CKD, we found a significant increase in LBP levels across the different stages of CKD (ANOVA, P: 0.001). When analyzed as a continuous variable, LBP is significantly inversely correlated with eGFR (P &amp;lt; 0.001; Spearman, r: −0.221), and positively correlated with CRP (P &amp;lt; 0.001; Spearman, r: 0.592). During a median follow-up of 56 (IQR, 53–59) months, 70 patients died, with more deaths observed among patients with LBP in higher tertiles [tertiles 1–3: 11, 22, and 37 events, respectively (Fig. 1)]. In univariate Cox proportional hazard analysis, plasma LBP was significantly associated with mortality [hazard ratio (HR) of 1.032; 95% confidence interval (95% CI): 1.014– 1.046; P &amp;lt; 0.001). This association remained significant in multivariate models with adjustment for age, sex and BMI. In two different rat models, induction of CKD resulted in a significant increase in LBP (P &amp;lt; 0.001). In these animals, we observed a significant inverse association between eGFR (and measured creatinine clearance) and LBP concentrations (P &amp;lt; 0.001; Spearman, r: −0.460). CONCLUSION Patients with CKD have higher levels of LBP, with higher levels of plasma LBP present in patients with more advanced CKD. Plasma LBP is an independent predictor of mortality. Experimentally induced CKD equally results in significantly increased LBP. Our data suggest that CKD leads to increased passage of LPS across the intestinal barrier. These findings strengthen the hypothesis of the gut–kidney axis as a source of chronic inflammation.