Inactivation Of Endotoxin Research Articles

Affinity and Inactivation of Bacterial Endotoxins for Medical Device Materials.

Endotoxins are high-molecular-weight complexes that contain lipopolysaccharide, protein, and phospholipid originating from the outer membrane of gram-negative bacteria. As gram-negative bacteria are naturally present in a variety of sources, endotoxins are commonly identified as contaminants in manufacturing environments. In industrial applications, endotoxin often is considered difficult to inactivate and to have a strong affinity with surfaces resulting from its hydrophobic chemical structure. This article describes the investigation of the true affinity of endotoxin, from various microbial sources in solution, for medical device material surfaces. In addition, endotoxin reduction was investigated with commonly used sterilization methods such as those based on ionizing radiation, dry and moist heat, and ethylene oxide sterilization. Endotoxin activity was found to be reduced following exposure to a range of sterilization modalities with the degree of activity reduction related to the source of endotoxin and the substrate material upon which it was present.

Inactivation of bacterial endotoxin in water using a pressurized microfluidic ultra-high temperature (UHT) pasteurizer

Ultra-pure water is critical for certain medical applications such as preparation of dialysis fluid and formulation of intravenous medications. However, production of ultra-pure water is challenging due to the difficulty of inactivating or removing endotoxins in water. The main objective of this study was to evaluate the ability of a pressurized microfluidic ultra-high temperature (UHT) pasteurizer to inactivate endotoxins (>2 log EU/ml) in water to meet the sterility assurance limit. A solution of Pseudomonas endotoxin (300 EU/ml) was prepared in endotoxin-free water and treated at various temperatures (150 °C–230 °C), pressures (1.65–3.10 MPa) and flow rates (100–400 ml/min). Endotoxin concentrations were determined using the Kinetic Turbidimetric LAL Assay. Endotoxin was effectively heat stable when treated at temperatures between 150 °C and 184 °C. Endotoxin inactivation at 200 °C was significant and was influenced by flow rate; however, even the slowest flow rate (100 ml/min) could not confidently achieve >2 log reduction of endotoxin. Increasing the temperature/pressure (230 °C/3.10 MPa) resulted in >3.8 log EU/ml reduction of endotoxin at all flow rates (100–400 ml/min). This is the first demonstration that microfluidic UHT pasteurization could be an effective process for inactivating endotoxin in water used in medical devices and for other continuous flow applications.

Engineering catalytic dephosphorylation reaction for endotoxin inactivation

Although lives have been saved due to the discovery of endotoxin removal methods including solvent extraction and affinity adsorption, they have limitations in treatment capacity, efficiency or costs. Endotoxin contaminations still result in a large number of deaths in global every year. This necessitates a mechanistic breakthrough for endotoxin removal or inactivation. Herein, we engineered a dephosphorylation reaction on endotoxins by a synthetic nanozyme (CeO2) to attenuate the toxicity. CeO2 prepared in phosphate-free hydrothermal reaction selectively and efficiently catalyzed the breaking of P-O bonds in endotoxins. Catalytic depletion of phosphates from endotoxins attenuated their binding with Toll-like receptors, NF-κB activation and pro-inflammatory cytokine release. Airborne LPS was, for the first time, inactivated (98%) by this facile dephosphorylation reaction. A CeO2 integrating column displayed a 16-fold higher treatment capacity than commercial resins to aqueous endotoxins (water and protein solution). Overall, our findings offer a different mechanistic insight for removal of endotoxins.

Human cholesteryl ester transfer protein lacks lipopolysaccharide transfer activity, but worsens inflammation and sepsis outcomes in mice

Bacterial lipopolysaccharides (LPSs or endotoxins) can bind most proteins of the lipid transfer/LPS-binding protein (LT/LBP) family in host organisms. The LPS-bound LT/LBP proteins then trigger either an LPS-induced proinflammatory cascade or LPS binding to lipoproteins that are involved in endotoxin inactivation and detoxification. Cholesteryl ester transfer protein (CETP) is an LT/LBP member, but its impact on LPS metabolism and sepsis outcome is unclear. Here, we performed fluorescent LPS transfer assays to assess the ability of CETP to bind and transfer LPS. The effects of intravenous (iv) infusion of purified LPS or polymicrobial infection (cecal ligation and puncture [CLP]) were compared in transgenic mice expressing human CETP and wild-type mice naturally having no CETP activity. CETP displayed no LPS transfer activity in vitro, but it tended to reduce biliary excretion of LPS in vivo. The CETP expression in mice was associated with significantly lower basal plasma lipid levels and with higher mortality rates in both models of endotoxemia and sepsis. Furthermore, CETPTg plasma modified cytokine production of macrophages in vitro. In conclusion, despite having no direct LPS binding and transfer property, human CETP worsens sepsis outcomes in mice by altering the protective effects of plasma lipoproteins against endotoxemia, inflammation, and infection.

Endotoxin and metabolically associated pathology

The article presents modern concepts on the role of the intestinal microbiome in the pathogenetic mechanisms of the development of metabolically associated diseases. The history of endotoxin discovery has been described. The multifaceted endotoxin properties have been established in relation to both infectious and non‑infectious pathology. A stress theory of the regulation of intestinal lipopolysaccharide intake into the general bloodstream is described, and research data supporting the hypothesis are presented. The data on the physiological role of lipopolysaccharide and data on its characteristics depending on age, stress and physical activity are presented. The main mechanisms of the development of endotoxemia are shown, including the role of the disturbance of eubiosis of the intestinal microbiome and an increase in intestinal permeability. The role of the liver in the primary inactivation of endotoxin in healthy individuals has been elucidated. The role of lipids in the transport of intestinal lipopolysaccharide is indicated. The concepts of «endotoxin aggression» and «metabolic endotoxin aggression» are described. The role of a high‑fat diet in the development of endotoxemia, including through the development of dysbiosis, was revealed. The results of experimental studies on the administration of lipopolysaccharide to laboratory animals are described — the administration of endotoxin induces an increase in the level of pro‑inflammatory cytokines. The mechanisms of its interaction with Toll‑like receptors and the consequences of this interaction expressed in the potentiation of chronic inflammation in the liver tissue are presented. The role of endotoxin aggression in the induction of low grade inflammation in obese patients and the relationship with changes in the intestinal microbiome have been disclosed. The effects of lipopolysaccharide in the development of insulin resistance and type 2 diabetes mellitus are shown. The data of studies on the role of endotoxinemia in the development of non‑alcoholic fatty liver disease with the specification of the mechanism of interaction of lipopolysaccharide with liver cells and the progression of its course with the development of steatohepatitis and fibrosis.

Development of innovative technology for inactivation of endotoxins at low temperature condition utilizing gas phase sterilization system

Background & Aim With the rapid development of gene and cell therapy, it is increasingly important to address the continuing risk concerns of endotoxins involving in the manufacturing and quality control of medical products and devices. Although dry-heat sterilization is generally considered as the preferred method for rendering heat-stable materials free of endotoxin, there are limitations to the effective approaches available to inactivate bacterial endotoxin, especially at low temperature. We have developed innovative technology as gas phase sterilization system based on a catalytic reaction mechanism by use of methanol to generate mixed biogas, exhibiting remarkable performance of nucleic acid decomposition as well as sterilization. Here we will present our recent challenge of findings of additional remarkable function of this system exhibiting inactivation of bacterial endotoxin at even low temperature around room temperature. Methods, Results & Conclusion Standard endotoxin indicators 2,000 EU/vial (2K-ET) (Charles River Laboratores, CRL) were used for the experiments, and endotoxin concentrations were determined by Endosafe®-PTS™ instrument including disposable PTS™ cartridge FDA (0.05-5 EU/mL) (CRL). 2K-ET vial was directory exposed to mixed biogas in apparatus for 60 min at 37 or 60 degree celsius, and endotoxin concentrations were measured using 2K-ET samples dissolved in 1mL endotoxin-free water. After biogas exposure for 60 min., endotoxin concentration levels showed remarkably reduction up to 6∼11 %, and those effects suggested to be temperature dependent; 83.3±4.7 EU/mL at 37 degree, 46.0±0.85 EU/mL at 60 degree, control sample (non-exposure): 725±23.5 EU/mL. Thus, this innovative gas phase sterilization system could exhibit valuable potential as practical performance of endotoxin inactivation at low temperature as 37 degree. Although it is still necessary to develop the reduction performance into 3-Log reduction, this advantageous function is expected to be widely useful and have high potential in the public health and pharmaceutical industries in the future.

Low-dose polymyxin: an option for therapy of Gram-negative sepsis.

Endotoxins are the major components of the outer membrane of most Gram-negative bacteria and are one of the main targets in inflammatory diseases. The presence of endotoxins in blood can provoke septic shock in case of pronounced immune response. Here we show in vitro inactivation of endotoxins by polymyxin B (PMB). The inflammatory activity of the LPS–PMB complex in blood was examined in vitro in freshly drawn blood samples. Plasma protein binding of PMB was determined by ultracentrifugation using membranes with different molecular cut-offs, and PMB clearance during dialysis was calculated after in vitro experiments using the AV1000S filter. The formed LPS–PMB complex has lower inflammatory activity in blood, which results in highly reduced cytokine secretion. According to in vitro measurements, the appropriate plasma level of PMB for LPS inactivation is between 100 and 200 ng/ml. Furthermore, the combination of cytokine removal by adsorbent treatment with LPS inactivation by PMB dosage leads to strong suppression of inflammatory effects in blood in an in vitro model. Inactivation of endotoxins by low-dose intravenous PMB infusion or infusion into the extracorporeal circuit during blood purification can be applied to overcome the urgent need for endotoxin elimination not only in treatment of sepsis, but also in liver failure.

Inactivation of Bacterial Spore, Endotoxin, Lipid A, Normal Prion and Abnormal Prion by Exposures to Several Sorts of Gases Plasma.

This review discusses the application of several sorts of non-equilibrium gas plasma discharges for sterilization and disinfection treatments against spores or bioburden on/in the healthcare products or biological indicators. The basic properties of electrical discharges are briefly reviewed and thereafter the paper discusses the interactions of gas plasma with several sorts of biological systems such as bacteria, bacterial spores, endotoxins, lipid A and normal and abnormal prion proteins.

Polymyxin-Coated Nanostructured Materials: An Option for Sepsis Treatment

Polymyxin-Coated Nanostructured Materials: An Option for Sepsis Treatment Objective: Endotoxins (lipopolysaccharides, LPS) are among the main targets in extracorporeal therapies. LPS, which is the major component of the outer cell wall of Gram-negative bacteria, strongly induces inflammatory responses in humans at concentrations lower than 1 ng/kg body weight. Although the elimination of LPS is promising for the supportive therapy of sepsis and liver failure, endotoxin neutralization using endotoxin adsorbents is controversial. Earlier studies show that Polymyxin B (PMB) could be applied for endotoxin inactivation in blood. Aim of this study was to establish an adsorbent-based PMB release system which ensures a constant PMB level in plasma during extracorporeal therapies. Methods: A polystyrene-divinylbenzene based cytokine adsorbent (CG161c) with nanostructured pores was coated with a defined amount of PMB by hydrophobic interactions. The PMB release by the PMB coated adsorbent was studied in plasma and fractionated plasma. Results: In plasma or blood, an equilibration between the free and bound form of PMB leads to a constant PMB level in plasma. The PMB release was influenced by the adsorbent inner surface area and the protein concentration of the plasma. In fractionated plasma where the protein concentration is lower, the PMB release was much less than in whole plasma. Additionally we could show that the PMB coating doesn´t influence the cytokine removal of the CG161c adsorbent. Conclusion: Our in vitro model shows that the combination of cytokine removal and controlled PMB release by the same adsorbent could be an option for Gram-negative sepsis treatment.

Inactivation of Bacterial Spore, Endotoxin, Lipid A, Normal Prion and Abnormal Prion by Nitrogen Gas Plasma Exposure

Inactivation of Bacterial Spore, Endotoxin, Lipid A, Normal Prion and Abnormal Prion by Nitrogen Gas Plasma Exposure

Photoactivated Polycationic Bioactive Chitosan Nanoparticles Inactivate Bacterial Endotoxins

IntroductionThe current root canal disinfection protocols fail to markedly inactivate bacterial endotoxins from infected root dentin. This study aimed to evaluate the ability of antibacterial photodynamic therapy with chitosan-conjugated rose bengal nanoparticles (CSRBnps) to selectively inactivate endotoxins/lipopolysaccharides (LPSs). MethodsAntimicrobial agents such as calcium hydroxide (Ca[OH]2), chitosan nanoparticles (CSnps), CSRBnps, and methylene blue (MB) were assessed for their ability to neutralize LPSs obtained from Pseudomonas aeruginosa in a time-dependent interaction with/without photoactivation (20 and 40 J/cm2). The inflammatory potential of the treated/untreated LPSs was assessed on macrophage cells (RAW 267.4) using nitric oxide– and enzyme-linked immunosorbent assay (tumor necrosis factor α and interleukin-6 expression)–based analysis. These antimicrobials were tested directly on macrophage cells for cytotoxicity using the mitochondrial activity assay and light microscopy. The data were analyzed using 1-way analysis of variance and the Tukey test. ResultsCSnps were least effective in LPS inactivation. Interluekin-6 expression was reduced only with CSRBnp treatment. CSnps and CSRBnps were completely nontoxic, and MB showed slight toxicity to macrophage cells. Ca(OH)2 was highly cytotoxic (P < .005) even at 30 minutes of exposure. CSRBnps and MB with/without photoactivation significantly inactivated LPSs with reduced nitric oxide and tumor necrosis factor α expression (P < .05). Cell death and detachment after Ca(OH)2 treatment resulted in complete absence of all 3 inflammatory markers. ConclusionsPhotodynamically activated CSRBnps caused significant inactivation of endotoxins and the subsequent reduction of all tested inflammatory markers from activated macrophages. Antimicrobial CSRBnps in combination with photodynamic therapy showed the potential to effectively inactivate bacterial endotoxins.

Inactivation of Endotoxin and Lipid A by Nitrogen Gas Plasma Exposure Running title: Inactivation Endotoxin and lipid A

The gases plasma from several different gases has shown a sporicidal activity. From these gases, nitrogen gas was most difficult to produce atomic nitrogen radicals. However, these radicals have a high energy, indicating that nitrogen gas plasma could be used to sterilize microorganisms and inactivate endotoxins and lipid A. The sterilization mechanism of nitrogen gas plasma may be the synergistic effect of free radicals such as (OH radical and NO radical) and metastables (N2 or O2 metastable). Thus, the target microorganisms such as bioburden were damaged by degradation, which resulted in death. The biological indicator BI used in this study was Geobacillus stearothermophilus ATCC 7953 at an initial population of 106 CFU/carrier. Sterility assurance level (SAL) of 10-6 was confirmed by using the BI. In addition endotoxins were successfully inactivated by nitrogen gas plasma expossure. More than 5 log reduction of endotoxins could be attained within 30 minutes of nitrogen gas plasma exposure. Lipid A degradation by nitrogen gas plasma exposure was confirmed by LC-MS-MS. Material/functional compatibility was also maintained by nitrogen gas plasma exposure due to shallower penetration depth of 10-20 nm. No deterioration of polymers could be observed by nitrogen gas plasma exposure. Residual gases, if any, are non-toxic. From the above description, it can be said that nitrogen gas plasma sterilization is an alternative future sterilization method.

Inactivation of Bacterial Spore, Endotoxin, Lipid A, and Abnormal Prion by Gas Plasma Exposure

Inactivation of Bacterial Spore, Endotoxin, Lipid A, and Abnormal Prion by Gas Plasma Exposure

Rapid point-of-use water purification using nanoscale zero valent iron (nZVI) particles

Nanoscale zero-valent iron (nZVI) particles are increasingly being investigated in removing aqueous contaminants. Here, we have demonstrated its inactivation and magnetic removal of bacteria and endotoxins from environmental wastewater samples. Varying dosages (10–1,000 μL) of 0–6 days aged nZVI with a concentration of 5 mg/mL for 2 mL wastewater samples were tested, and relevant removal efficiencies were determined using culturing method for bacteria and limulus amebocyte lysate (LAL) for endotoxins. The supernatants of wastewater samples after reacting with nZVI and subsequent magnetic separations were subjected to spectroscopic, qPCR and DGGE analysis. Overall, high magnetic bacterial removal efficiencies were observed up to 3–4 logs for 1 mL nZVI, while the removal efficiencies decreased sharply down to 0.5 log for 10 μL nZVI. qPCR and DGGE results revealed that higher dosages of nZVI caused severe bacterial cell membrane ruptures, releasing significant amounts of DNA up to 107–108 gene copies/mL when 1 mL nZVI was used. Richer DGGE patterns were observed for higher nZVI dosages. In addition, regardless of the dosages (10–1,000 μL) we have observed more than 90 % removal of endotoxins from the wastewater samples. The described technology has great promise to be used as a point-of-use water purification solution for various purposes.

Endotoxin inactivation via steam‐heat treatment in dilute simethicone emulsions used in biopharmaceutical processes

Simethicone emulsion is used to regulate foaming in cell culture operations in biopharmaceutical processes. It is also a potential source of endotoxin contamination. The inactivation of endotoxins in dilute simethicone emulsions was assessed as a function of time at different steam temperatures using a Limulus amebocyte lysate kinetic chromogenic technique. Endotoxin inactivation from steam-heat treatment was fit to a four-parameter double exponential decay model, which indicated that endotoxin inactivation was biphasic, consisting of fast and slow regimes. In the fast regime, temperature-related effects were dominant. Transitioning into the slow regime, the observed temperature dependence diminished, and concentration-related effects became increasingly significant. The change in the Gibbs free energy moving through the transition state indicated that a large energy barrier must be overcome for endotoxin inactivation to occur. The corresponding Arrhenius pre-exponential factor was >>10(12) s(-1) suggesting that endotoxins in aqueous solution exist as aggregates. The disorder associated with the endotoxin inactivation reaction pathway was assessed via the change in entropy moving through the transition state. This quantity was positive indicating that endotoxin inactivation may result from hydrolysis of individual endotoxin molecules, which perturbs the conformation of endotoxin aggregates, thereby modulating the biological activity observed. Steam-heat treatment decreased endotoxin levels by 1-2 logarithm (log) reduction (LRV), which may be practically relevant depending on incoming raw material endotoxin levels. Antifoam efficiency and cell culture performance were negligibly impacted following steam-heat treatment. The results from this study show that steam-heat treatment is a viable endotoxin control strategy that can be implemented to support large-scale biopharmaceutical manufacturing.

Experimental Study on Inactivation of Bacterial Endotoxin by Using Dielectric Barrier Discharge

The low-temperature plasma (LTP) generated by dielectric barrier discharge (DBD) was used to sterilize the E.coli endotoxin, which is usually difficult to kill by traditional methods. Three different concentrations of bacterial endotoxin (1 EU/mL, 0.5 EU/mL and 0.25 EU/mL) were treated by LTP for different time (20 s, 40 s and 60 s). Tachypleus amebocyte lysate (TAL) method was employed to detect the concentration variation of bacterial endotoxin before and after the plasma treatment, and endotoxic shock mice model was used to evaluate the inactivation effects of LTP on endotoxin for further study. Experimental results demonstrated that, DBD plasma can inactivate the bacterial endotoxin quickly and effectively, and when the LTP treatment time was increased, the concentrations of bacterial endotoxin decreased gradually (after 60 s plasma treatment, its inactivation effect was beyond the Chinese pharmacopoeia standard), and the average survival time of mice gradually extended. The possible inactivation mechanisms are proposed to be related to reactive oxygen species (ROSs).

Kinetics of Hydrothermal Inactivation of Endotoxins

A kinetic model was established for the inactivation of endotoxins in water at temperatures ranging from 210°C to 270°C and a pressure of 6.2 × 10(6) Pa. Data were generated using a bench scale continuous-flow reactor system to process feed water spiked with endotoxin standard (Escherichia coli O113:H10). Product water samples were collected and quantified by the Limulus amebocyte lysate assay. At 250°C, 5-log endotoxin inactivation was achieved in about 1 s of exposure, followed by a lower inactivation rate. This non-log-linear pattern is similar to reported trends in microbial survival curves. Predictions and parameters of several non-log-linear models are presented. In the fast-reaction zone (3- to 5-log reduction), the Arrhenius rate constant fits well at temperatures ranging from 120°C to 250°C on the basis of data from this work and the literature. Both biphasic and modified Weibull models are comparable to account for both the high and low rates of inactivation in terms of prediction accuracy and the number of parameters used. A unified representation of thermal resistance curves for a 3-log reduction and a 3 D value associated with endotoxin inactivation and microbial survival, respectively, is presented.

Inactivation of Escherichia coli endotoxin by soft hydrothermal processing.

Bacterial endotoxins, also known as lipopolysaccharides, are a fever-producing by-product of gram-negative bacteria commonly known as pyrogens. It is essential to remove endotoxins from parenteral preparations since they have multiple injurious biological activities. Because of their strong heat resistance (e.g., requiring dry-heat sterilization at 250 degrees C for 30 min) and the formation of various supramolecular aggregates, depyrogenation is more difficult than sterilization. We report here that soft hydrothermal processing, which has many advantages in safety and cost efficiency, is sufficient to assure complete depyrogenation by the inactivation of endotoxins. The endotoxin concentration in a sample was measured by using a chromogenic limulus method with an endotoxin-specific limulus reagent. The endotoxin concentration was calculated from a standard curve obtained using a serial dilution of a standard solution. We show that endotoxins were completely inactivated by soft hydrothermal processing at 130 degrees C for 60 min or at 140 degrees C for 30 min in the presence of a high steam saturation ratio or with a flow system. Moreover, it is easy to remove endotoxins from water by soft hydrothermal processing similarly at 130 degrees C for 60 min or at 140 degrees C for 30 min, without any requirement for ultrafiltration, nonselective adsorption with a hydrophobic adsorbent, or an anion exchanger. These findings indicate that soft hydrothermal processing, applied in the presence of a high steam saturation ratio or with a flow system, can inactivate endotoxins and may be useful for the depyrogenation of parenterals, including end products and medical devices that cannot be exposed to the high temperatures of dry heat treatments.

Effect of lipid-bound apoA-I cysteine mutants on lipopolysaccharide-induced endotoxemia in mice

HDL has been shown to be able to neutralize the toxicity of lipopolysaccharide (LPS). Our previous study (J. Lipid Res. 2005. 46: 1303-1311) characterized the properties of secondary structure and in vitro functions of different cysteine mutants of apolipoprotein A-I. Here, we reconstituted recombinant HDLs (named rHDLwt, rHDL52, rHDL74, rHDL107, rHDL129, rHDL173, rHDL195, and rHDL228) by mixing wild type or those mutants with dipalmitoyl phosphatidylcholine and examined their in vivo effects on LPS-induced endotoxemia in mice. Our results showed that 24 h after injection, mice receiving rHDL74 or rHDL52 had a significant decrease of plasma tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta), compared with control mice receiving either saline or rHDLwt (P < 0.05). Administration of rHDL74 to mice injected with LPS also led to a decrease of plasma IL-6, protection of lung against acute injury, and attenuation of endotoxin-induced clinical symptoms in mice, compared with controls injected with LPS only. However, injection of rHDL228 significantly increased plasma concentration of TNF-alpha and exacerbated LPS-induced lung injury. In summary, compared with rHDLwt, rHDL74 and rHDL52 exhibit higher anti-inflammation capabilities, whereas rHDL228 shows hyper-proinflammation by exacerbating LPS-induced endotoxemia in mice.

Effect of different methods of sterilization on the inactivation of bacterial endotoxin (LPS) in endodontic files

This study evaluated the presence of lipopolysaccharide (LPS) in endodontic files after in vitro contamination with E. coli LPS and sterilization in dry heat or wet heat, with or without previous immersion in a Ca(OH)2 suspension. LPS was quantified using the Kinetic QCLTM test and data were analyzed statistically by the t-test. LPS was quantified only in the contaminated group, not submitted to any immersion or sterilization procedure (0.262±0.1296 endotoxin units/mL) (p=0.0003). In conclusion, both wet heat and dry heat sterilization were effective in inactivating LPS without the need of previous immersion of the files in a Ca(OH)2 suspension.