Amounts Of Lipopolysaccharide Research Articles

Lipopolysaccharide with long O-antigen is crucial for Salmonella Enteritidis to evade complement activity and to facilitate bacterial survival in vivo in the Galleria mellonella infection model

Bacterial resistance to serum is a key virulence factor for the development of systemic infections. The amount of lipopolysaccharide (LPS) and the O-antigen chain length distribution on the outer membrane, predispose Salmonella to escape complement-mediated killing. In Salmonella enterica serovar Enteritidis (S. Enteritidis) a modal distribution of the LPS O-antigen length can be observed. It is characterized by the presence of distinct fractions: low molecular weight LPS, long LPS and very long LPS. In the present work, we investigated the effect of the O-antigen modal length composition of LPS molecules on the surface of S. Enteritidis cells on its ability to evade host complement responses. Therefore, we examined systematically, by using specific deletion mutants, roles of different O-antigen fractions in complement evasion. We developed a method to analyze the average LPS lengths and investigated the interaction of the bacteria and isolated LPS molecules with complement components. Additionally, we assessed the aspect of LPS O-antigen chain length distribution in S. Enteritidis virulence in vivo in the Galleria mellonella infection model. The obtained results of the measurements of the average LPS length confirmed that the method is suitable for measuring the average LPS length in bacterial cells as well as isolated LPS molecules and allows the comparison between strains. In contrast to earlier studies we have used much more precise methodology to assess the LPS molecules average length and modal distribution, also conducted more subtle analysis of complement system activation by lipopolysaccharides of various molecular mass. Data obtained in the complement activation assays clearly demonstrated that S. Enteritidis bacteria require LPS with long O-antigen to resist the complement system and to survive in the G. mellonella infection model.

Clinical and microbiological effects of ultrasonically activated irrigation versus syringe irrigation during endodontic treatment: a systematic review and meta-analysis of randomized clinical trials.

This study aimed to systematically review clinical and microbiology-related effects of ultrasonically activated irrigation (UAI) compared to syringe irrigation (SI) during endodontic treatment. Electronic databases searching and manual searching were conducted. Only randomized clinical trials (RCTs) were included comparing UAI to SI. The RoB 2.0 Cochrane tool was used for risk-of-bias (RoB) assessment. The main outcomes were postoperative pain, treatment failure, and microbiology-related outcomes. Qualitative and quantitative analyses, wherever applicable, were performed. Risk ratios (RR) and [standardized] mean differences {[S]MD} were calculated for dichotomous and continuous outcomes, respectively. Certainty of evidence (CoE) was assessed using GRADE tool. Ten RCTs were included. UAI reduced pain incidence within the first 24h (RR 0.50, 95% CI 0.35-0.71, 308 teeth) and microbial counts (SMDpooled -0.40, 95% CI [-0.78, -0.02], I2 = 0%, 126 teeth) than SI in non-vital teeth with apical periodontitis (AP). Both groups, however, had similar effects regarding pain intensity, lipopolysaccharide amounts, and the incidence of rescue-analgesic intake, treatment failure, and microbial presence (p > 0.05). CoE ranged from low to very low. Very limited evidence suggests that UAI could reduce postoperative-pain risk within the first 24h and microbial counts for non-vital teeth with AP compared to SI. Most meta-analyses, however, are based on very few studies, mostly low-powered, with an overall very-low-to-low CoE. Further well-designed, larger RCTs are, thus, required.

산업폐수로부터 분리한 1,4-Dioxane을 분해할 수 있는 새로운 세균인 Shinella granuli CK-4의 특성

We have characterized a novel bacterium, strain CK-4 isolated from industrial wastewater, which is capable of utilizing 1,4-dioxane as sole carbon source and energy. Complete depletion of 1 g/L 1,4-dioxane by CK-4 cultures growing on AMS media was achieved within 168 h. Both the Biolog system and phylogenetic analysis using 16S rRNA sequencing were utilized for identification, and the strain was designated as Shinella granuli CK-4, and registered in GenBank as [MN841914]. The mortality of CK-4 was not greatly affected by 1,4-dioxane concentrations below 10 g/L but was increased with increasing 1,4-dioxane concentrations. SDS-PAGE revealed that the amount of lipopolysaccharide in CK-4 increased or decreased according to the 1,4-dioxane concentration. The expression of 60-kDa GroEL stress shock protein (SSP) was increased in proportion to concentrations below 10 g/L 1,4-dioxane but was not expressed at concentrations above 15 g/L dioxane. On the other hand, 70-kDa DnaK SSP was not expressed at any 1,4-dioxane concentrations (0~20 g/mL) tested. SEM demonstrated that the surface of CK-4 cells exposed to 10 g/L dioxane was not significantly different from that of normal cells, whereas cells exposed to 20 g/L 1,4-dioxane showed the presence of perforations and irregular rod forms with wrinkled cell surfaces. These results provide clues to understanding the resistance of S. granuli CK-4 to 1,4-dioxane and the promotion of 1,4-dioxane degradation, which can be applied to the treatment of industrial wastewater with higher concentrations of 1,4-dioxane.

COVID-19 War, Human Microbiota Function

COVID-19 War, Human Microbiota Function

Melimine-Coated Antimicrobial Contact Lenses Reduce Microbial Keratitis in an Animal Model

To determine the ability of antimicrobial peptide melimine-coated contact lenses to reduce the incidence of microbial keratitis (MK) in a rabbit model of contact lens wear. In vitro antimicrobial activity of melimine-coated contact lenses was determined against Pseudomonas aeruginosa by viable count and a radiolabeled assay. The amount of lipopolysaccharide (LPS) associated with bacteria bound to melimine-coated and control lenses was determined. Ocular swabs from rabbit eyes were collected for assessment of ocular microflora. A rabbit model for MK was developed that used overnight wear of contact lenses colonized by P. aeruginosa in the absence of a corneal scratch. During lens wear, detailed ocular examinations were performed, and the incidence of MK was investigated. Bacteria associated with worn lenses and infected corneas were determined by viable plate count. Inhibition in viable and total P. aeruginosa adhesion by melimine-coated contact lenses was 3.1 log10 and 0.4 log10, respectively. After colonization, the amount of LPS on lenses was approximately the same with or without melimine. Gram-positive bacteria were found in all the ocular swabs followed by fungus (42%). Melimine-coated lens wear was protective and significantly (odds ratio 10.12; P = 0.012) reduced the incidence of P. aeruginosa-driven MK in the rabbit model. The antimicrobial lenses were associated with significantly (P < 0.001) lower ocular scores, indicating improved ocular signs compared with controls. This study showed that contaminated contact lenses can produce MK without corneal epithelial defect in an animal model. Melimine-coated contact lenses reduced the incidence of MK associated with P. aeruginosa in vivo. Development of MK requires viable bacteria adherent to contact lenses, and bacterial debris adherent at the lens surface did not cause keratitis.

GSK2656157, a PERK inhibitor, reduced LPS-induced IL-1β production through inhibiting Caspase 1 activation in macrophage-like J774.1 cells

IL-1β is one of the inflammatory cytokines and is cleaved from pro-IL-1β proteolytically by activated Caspase 1. For the activation of Caspase 1, inflammasome was formed by two signals, what is called, priming and triggering signals. In this study, it was found that mouse macrophage J774.1 cells, when treated by single large amount of lipopolysaccharide (LPS), produced a significant amount of IL-1β. On the other hand, IL-1β production was not detected when treated by a single, small amount of LPS. Then, focusing on endoplasmic reticulum (ER) stress response among stress responses induced by a large amount of LPS, when GSK2656157, a PERK inhibitor, was used for inhibition of ER stress, GSK2656157 reduced IL-1β production dose-dependently. Next, when Thapsigargin, an ER stress reagent, was added with LPS, IL-1β production increased more than by LPS alone. Thus, these results suggested that ER stress was involved in LPS-induced IL-1β production. When the activation of Caspase 1 was examined by fluorescence activated cell sorter analysis, it was found that GSK2656157 inhibited LPS-induced Caspase 1 activation. Further, it was confirmed that GSK2656157 did not affect LPS-induced TNF-α production and activation of NF-κB and specifically inhibited the PERK/eIF-2α pathway. Therefore, it was found that GSK2656157 specifically inhibited ER stress induced by large amount of LPS and reduced LPS-induced IL-1β production through inhibition of Caspase 1 activation.

Effects of two Asian sand dusts transported from the dust source regions of Inner Mongolia and northeast China on murine lung eosinophilia

The quality and quantity of toxic materials adsorbed onto Asian sand dust (ASD) are different based on dust source regions and passage routes. The aggravating effects of two ASDs (ASD1 and ASD2) transported from the source regions of Inner Mongolia and northeast China on lung eosinophilia were compared to clarify the role of toxic materials in ASD. The ASDs contained different amounts of lipopolysaccharides (LPS) and β-glucan (ASD1<ASD2) and SiO2 (ASD1>ASD2). CD-1 mice were instilled intratracheally with ASD1, ASD2 and/or ovalbumin (OVA) four times at 2-week intervals. ASD1 and ASD2 enhanced eosinophil recruitment induced by OVA in the submucosa of the airway, with goblet cell proliferation in the bronchial epithelium. ASD1 and ASD2 synergistically increased OVA-induced eosinophil-relevant cytokines interleukin-5 (IL-5), IL-13 (ASD1<ASD2) and chemokine eotaxin (ASD1>ASD2) in bronchoalveolar lavage fluid. ASD2 aggravating effects on lung eosinophilia were greater than ASD1. The role of LPS and β-glucan in ASD2 on the production of pro-inflammatory mediators was assessed using in vitro bone marrow-derived macrophages (BMDMs) from wild type, Toll-like receptor 2-deficient (TLR2−/−), TLR4−/−, and MyD88−/− mice (on Balb/c background). ASD2-stimulated TLR2−/− BMDMs enhanced IL-6, IL-12, TNF-α, MCP-1 and MIP-1α secretion compared with ASD2-stimulated TLR4−/− BMDMs. Protein expression from ASD2-stimulated MyD88−/− BMDM were very low or undetectable. The in vitro results indicate that lung eosinophilia caused by ASD is TLR4 dependent. Therefore, the aggravation of OVA-related lung eosinophilia by ASD may be dependent on toxic substances derived from microbes, such as LPS, rather than SiO2.

Analysis of Soluble Protein Complexes in Shigella flexneri Reveals the Influence of Temperature on the Amount of Lipopolysaccharide

Shigella flexneri, which is closely related to Escherichia coli, is the most common cause of the endemic form of shigellosis. In this study, 53 homomultimeric protein complexes and nine heteromultimeric protein complexes from S. flexneri 2a strain 2457T were separated and identified. Among these, three potential homomultimeric protein complexes had not been previously described. One complex, thought to be composed of 12 PhoN1 subunits, is a periplasmic protein with an unknown physiological role encoded on the virulence plasmid of S. flexneri. The abundance of the protein complexes was compared following growth at 37 or 30°C, and the abundance of three protein complexes (PyrB-PyrI, GlmS, and MglB) related to the synthesis of lipopolysaccharides (LPS) appeared to be temperature-dependent. Many studies have shown that LPS is essential to the virulence of S. flexneri. Here, we report the influence of temperature on the amount of LPS.

Cellular and proteomic responses of Escherichia coli JK-17 exposed to the Rosa hybrida flower extract

The purpose of this work was to characterize the cellular and proteomic responses of Escherichia coli JK-17 exposed to the rose flower extract (Rosa hybrida). The bacterial isolate was enriched and isolated from contaminated food. 16S rRNA sequence analyses revealed that the strain was 99% similar to the E. coli species cluster; therefore, this strain was designated E. coli JK-17. The rose flower extract showed a dose-dependent antibacterial effect on E. coli JK-17. Treatment of E. coli JK-17 with 50 and 100 mg/mL of the rose flower extract completely inhibited growth within 12 and 6 h of incubation. The stress shock proteins (SSPs) were induced with different concentrations of rose flower extract. The proteins were identified as 70-kDa DnaK and 60-kDa GroEL by SDS-PAGE and Western blot using anti-DnaK and anti-GroEL monoclonal antibodies. The levels of SSPs induced by the rose flower extract increased when the exposure time to the rose flower extract was increased. SDS-PAGE with silver staining revealed that the amount of lipopolysaccharide (LPS) in E. coli JK-17 increased or decreased with different concentrations and exposure times of the rose flower extract. To identify proteins induced by the rose flower extract, 2-dimensional electrophoresis (2-DE) was applied to soluble protein fractions of E. coli JK-17 cultures. In the pH range of 4 ∼ 7, more than 250 spots were detected on the silver stained gels. Notably, 15 protein spots were increased or decreased after treatment with the rose flower extract. Twelve up-regulated proteins were identified as chaperones (DnaK and GroEL) and porin proteins (PhoE, RfaI, RfaG, MdoH, and WzzE) by MALDITOF mass spectrometry, and three down-regulated proteins were identified, including proteins involved in energy and DNA metabolism (SdhA and GyrB), and amino acid biosynthesis (GltK). Using scanning electron microscopic analysis, some cells were shown to adopt irregular rod shapes and wrinkled surfaces after treatment with the rose flower extract. These results provide clues for better understanding the mechanism of rose flower extract-induced stress and cytotoxicity in E. coli JK-17.

Growth-phase regulation of lipopolysaccharide O-antigen chain length influences serum resistance in serovars of Salmonella

The amount of lipopolysaccharide (LPS) O antigen (OAg) and its chain length distribution are important factors that protect bacteria from serum complement. Salmonella enterica serovar Typhi produces LPS with long chain length distribution (L-OAg) controlled by the wzz gene, whereas serovar Typhimurium produces LPS with two OAg chain lengths: an L-OAg controlled by Wzz(ST) and a very long (VL) OAg determined by Wzz(fepE). This study shows that serovar Enteritidis also has a bimodal OAg distribution with two preferred OAg chain lengths similar to serovar Typhimurium. It was reported previously that OAg production by S. Typhi increases at the late exponential and stationary phases of growth. The results of this study demonstrate that increased amounts of L-OAg produced by S. Typhi grown to stationary phase confer higher levels of bacterial resistance to human serum. Production of OAg by serovars Typhimurium and Enteritidis was also under growth-phase-dependent regulation; however, while the total amount of OAg increased during growth, the VL-OAg distribution remained constant. The VL-OAg distribution was primarily responsible for complement resistance, protecting the non-typhoidal serovars from the lytic action of serum irrespective of the growth phase. As a result, the non-typhoidal species were significantly more resistant than S. Typhi to human serum. When S. Typhi was transformed with a multicopy plasmid containing the S. Typhimurium wzz(fepE) gene, resistance to serum increased to levels comparable to the non-typhoidal serovars. In contrast to the relevant role for high-molecular-mass OAg molecules, the presence of Vi antigen did not contribute to serum resistance of clinical isolates of serovar Typhi.

THE INDUCTION OF SUPER-RESISTANCE USING SYNTHETIC LIPOPOLYSACCHARIDE RECEPTOR AGONIST RESCUES FATAL ENDOTOXEMIA IN RATS WITHOUT EXCESSIVE IMMUNOSUPPRESSION

Endotoxin tolerance provides protection against mortality under various conditions of stress. However, the induction of endotoxin tolerance thus far has no clinical application because of endotoxin toxicity and the excessive immune suppression that follows the tolerance induction. In this study, we examined whether a novel, synthetic lipopolysaccharide (LPS) receptor agonist, ER-803058 (ER) can induce endotoxin tolerance with accompanying low toxicity. The stimulative effects of ER on tumor necrosis factor (TNF)-alpha production from RAW264 cells were 50% to 70% lower than those of the corresponding quantities of LPS. ER pretreatment also diminished TNF-alpha secretion induced by a subsequent LPS shock. However, the degree of desensitization with ER pretreatment (10 ng/mL, 55.5% +/- 6.7%; 100 ng/mL, 42.3 +/- 4.9%) was modest in contrast with that measured for the corresponding LPS pretreatment (10 ng/mL, 36.7% +/- 3.7%; 100 ng/mL, 20.0% +/- 3.6%). The minimum in vivo dose (0.02 mg/kg/body weight) of ER-induced negligible production of TNF-alpha and interleukin (IL)-6 in rats, and resulted in a modest endotoxin tolerance with respect to TNF-alpha secretion. Although the plasma TNF-alpha level after ER pretreatment was decreased (48.2% +/- 1.1%), the suppression was not statistically significant. Interestingly, even this minimal quantity of ER pretreatment evoked a dramatic improvement in survival (90% survival) against administration of a lethal dose of LPS, which is inconsistent with the modest TNF-alpha suppression. Furthermore, ER pretreatment preserved normal plasma albumin levels and prevented the increase of plasma blood urea nitrogen levels seen with LPS. These results indicate that pretreatment with ER can effectively induce endotoxin tolerance, with a consequent improvement in mortality without toxicity and without subsequent excessive immunosuppression.

Endotoxin inhibitor blocks heat exposure-induced expression of brain cytokine mRNA in aged rats

To investigate the age-related changes in the expression of interleukin-1β (IL-1β) and its related substances in the brain during heat stress, we measured amounts of mRNAs for IL-1β, cyclooxygenase-2 (COX-2), and an inhibitor of nuclear factor (NF)-κB-β (IκB-β) that is known to reflect an activation of NF-κB, in the cortex, cerebellum, and hippocampus using a quantitative real-time capillary PCR method. The basal levels of IL-1β mRNA in aged rats (108–110 weeks old) was significantly higher than those in young animals (10–11 weeks old) in these brain regions. Heat exposure (33 °C) for 1 h enhanced the expression of IL-1β and COX-2 mRNAs in aged rats but not in young ones. The amount of lipopolysaccharide (LPS) assessed by its bioactivity in the cortex increased by heat exposure only in aged rats. To further examine an involvement of LPS in the increase in mRNAs, an endotoxin inhibitor (EI), a synthetic peptide that detoxifies LPS by binding to the toxic component of LPS, lipid A, was intraperitoneally injected before heat exposure in aged rats. An intraperitoneal injection of EI significantly attenuated the heat exposure-induced increases in mRNAs for IL-1β, COX-2, IκB-β, and the LPS activity. Administration of EI also debilitated the heat exposure-induced hyperthermia and responses of plasma ACTH and catecholamines. These findings, taken together, suggest that the bacterial translocation is involved in the mechanisms of the responses to heat exposure in aged rats including the increased expression of mRNAs for IL-1β and its related substances in the brain.

Rapid detection of a minute amount of lipopolysaccharide from Salmonella in large volumes by poly(ethyleneimine)-cloth enzyme immunoassay

Utilizing the rapidity in capturing Salmonella lipopolysaccharide (LPS) and the macroporous nature of poly(ethyleneimine) immobilized polyester cloth (PEI-cloth), a PEI-cloth disk set in a Swinnex cartridge joined at both ends with 10 ml plastic syringes was repeatedly passed through with an LPS sample in large volumes. The captured LPS on the disk were detected at up to 1 pg, which is equivalent to the amount produced of 16 cells, by cloth enzyme immunoassay.

Survival ofSerratia marcescensin the Presence of Complement

Serratia marcescens has been isolated from a variety of ocular infections and/or infammation. In order to survive in the eye, strains must be able to resist host defence systems. Complement has been shown to have an increased concentration and to be active during sleep. Not only is the complement system involved in host defence, but its activation can also produce in ammation. The aim of the present study was to examine the ability of ocular isolates of S. marcescens to withstand complement attack, activate complement and to examine the mechanisms used for evasion of this host defence system. The majority of isolates tested (4/6) were resistant to complement attack. These isolates were able to activate both complement pathways, as detected by enzyme-linked immunosorbent assays (ELISA) and western blotting assays. Components detected included C4, Factor B, C3 and C5. Possible mechanisms of resistance were elucidated including quantity of lipopolysaccharide (LPS) on the cell surface, ability to bind lactoferrin, types of complement components deposited onto the surfaces of strains, and the amount and location of C3 bound on the surface, as determined by transmission electron microscopy. Complement-resistant strains generated iC3b, bound more lactoferrin (1.7 ± 0.6 for resistant cells compared with 0.4 ± 0.2 for complement-sensitive cells, relative values), and had larger amounts of extractable endotoxin (216.8 ± 88.8 µ g/ml for resistant strains and 36.0 ± 48.4 µ g/ml for sensitive strains). Although there was no direct correlation between the ability to resist and activate complement and the isolation of strains from symptomatic or asymptomatic eyes, the resistance of strains to complement may allow these strains to remain in the eye and the activation of complement may contribute to the in ammatory response seen during infection or ocular infammation. Keywords: Serratia marcescens , complement, host defence, tear ?film.

Alteration of lipopolysaccharide and protein profiles in SDS-PAGE of rhizobia by osmotic and heat stress

The effects of osmotic and heat stress on lipopolysaccharides and proteins of rhizobia isolated from the root nodules of leguminous trees grown in semi-arid soils of the Sudan, and of agricultural legumes grown in salt-affected soils of Egypt, were determined by SDS-PAGE. The rhizobia were of three types: (1) sensitive strains, unable to grow in 3% (w/v) NaCl in yeast mannitol medium; (2) tolerant strains which could grow in 3% (w/v) NaCl; and (3) halophytic strains which grew with 3 to 10% (w/v) NaCl. The sensitive strains changed their gel pattern or the amount of lipopolysaccharide they synthesized when grown in 1% (w/v) NaCl. The tolerant and halophytic strains often modified their lipopolysaccharides in 3% NaCl, which was evident by a shift in the banding patterns towards longer chain length. Similar effects were observed in cells incubated with sucrose and, to a lesser extent, in cells incubated at growth temperatures near the recorded maximum temperature for growth. The stress-induced changes in lipopolysaccharides were not associated with specific banding patterns of the lipopolysaccharides. During incubation in medium containing elevated concentrations of NaCl or sucrose, the protein patterns of the rhizobia were also changed. A protein with relative mobility of 65 kDa appeared during temperature stress. The maximum growth temperature of the Sudanese rhizobia were up to 44.2°C.

Network of cytokine and arachidonic acid cascade in acute hepatic failure.

When heat-killed Propionibacterium acnes (P. acnes) was intravenously injected into mice and 7 days later a small amount of lipopolysaccharide (LPS) endotoxin was administered, most of the mice died of massive liver necrosis. In this liver injury model, cytokines, immunomediators, and eicosanoids, inflammatory products, were produced by Kupffer cells and liver-infiltrated macrophages, which were thought to participate, directly or indirectly, in the induction of liver cell damage. Furthermore, these two networks seemed to regulate each other. Thus, this regulatory mechanism might play an important role in the induction of liver cell injury.

Exposure effects of netilmicin on Enterobacter cloacae clinical isolates.

The effects of netilmicin exposure on two aminoglycoside (AMG)-resistant and two AMG-susceptible Enterobacter cloacae isolates were studied. It was found that 1 h incubation of bacterial suspensions with netilmicin at 16 mg/l reduces the amounts of lipopolysaccharides present on the bacterial surface. Also, netilmicin pretreatment increased both the susceptibility to normal human serum and the adhesion to human epithelial cells of all four E. cloacae strains examined.

Human Monocytes in a Long-Term Culture with Interleukin-2 Show High Tumoricidal Activity Against Various Tumor Cells

We compared the tumoricidal activity of human monocytes cultured with interleukin-2 (IL-2) or human recombinant interferon-gamma (IFN-gamma) alone, or IFN-gamma in combination with a small amount of lipopolysaccharides (LPS). Human monocytes cultured with IL-2 for 7 days or longer, termed lymphokine-activated macrophages (LAMs), showed higher tumoricidal activity than those cultured for 1 day. In contrast, monocytes cultured with IFN-gamma alone or in combination with LPS for 7 days or longer showed lower tumoricidal activity. LAMs were identified as macrophages by nonspecific esterase staining and immunofluorescence staining with anti-CD14 antibody. LAMs were not induced in fetal calf serum-containing medium, but they were induced when colony-stimulating factor-1 was added to the medium. LAMs showed high tumoricidal activity against all human and murine tumor cell lines tested, although they showed no cytotoxic activity against human normal cells. During incubation with IL-2, tumoricidal activity of LAMs was maximal at days 8-16 and was sustained until day 28. The difference in tumoricidal mechanism between LAMs and lymphokine-activated killer (LAK) cells was also shown by using two kinds of cytotoxic assay systems. LAMs require a long incubation time to kill tumor cells, but LAK cells can kill them immediately. Furthermore, LAMs kill tumor cells with complete DNA degradation, whereas LAK cells can induce significant but not complete DNA degradation. These results indicate that LAMs and LAK cells have different tumoricidal mechanisms for killing target cells, although they were induced by incubation with the same lymphokine, IL-2.

A new assay for bacterial lipopolysaccharides

The purpose of this study was to ascertain whether an assay for bacterial lipopolysaccharide (LPS) could be developed based on the specific, stoichiometric binding between Polymyxin B and lipopolysaccharides. Polymyxin B was covalently linked to Sepharose 4B and the LPS-binding capacity of the resulting gel determined by incubation with various amounts of radiolabeled LPS (14C-LPS) of known specific activity. Competitive binding studies showed an inverse linear relationship between the amount of unlabeled LPS added and the binding of14C-LPS. The assay enabled the detection of microgram quantities of LPS even though the specific activity of the14C-LPS used was very low. The study demonstrated the feasibility of a Polymyxin B-based assay for LPS, and possible ways in which the sensitivity of the assay may be increased are discussed.

Effects of bile acids on liver cell injury by cultured supernatant of activated liver adherents cells.

When heat-killed Propionibacterium acnes (P. acnes) is intravenously injected into mice followed by an intravenous injection of a small amount of lipopolysaccharide (LPS) 7 days later, most of the mice die of massive hepatic cell necrosis within 24 hours of LPS injection. In addition, when the liver adherent cells including Kupffer cells are separated from the mice 7 days after P. acnes injection and incubated in vitro with LPS, remarkable activity of the cytotoxic factor is found in the culture supernatant. This cytotoxic factor is thought to cause liver injury. Using this experimental model, the effects of various bile acids on liver cell injury were studied. As a result, ursodeoxycholic acid and dehydrocholic acid suppressed liver cell injury induced by the cytotoxic factor. However, cholic acid, deoxycholic acid and chenodeoxycholic acid did not have any hepatocytoprotective effects.