Endotoxin Contamination and Reaction Interfering Substances in the Plant Extract Library.

Endotoxin is an unintentional contaminant that has numerous activities and can affect various biological experiments using cells. In this study, we measured the endotoxin activity of samples from a plant extract library (PEL) and determined their degrees of contamination. Endotoxin was detected in approx. 48% (n = 139) and approx. 4% (n = 5) of field-collected and crude drug samples, respectively, and in concentrations >5.0 EU/mL in some samples. The concentrations of endotoxin that affect cells in vitro vary depending on the target cell type. Although the degree of contamination varied in the present study, it was considered to have little effect on the cell experiments. More than 150 PEL samples had problems with reaction courses or recovery rates of Limulus amoebocyte lysate (LAL) tests. In the LAL tests, using three plant extracts [Sanguisorba officinalis L. (Rosaceae), Oenothera biennis L. (Onagraceae), and Lythrum salicaria L. (Lythraceae)], the polyphenolic compounds in the plant extracts affected LAL test and their effects differed depending on the plant species. When the 16 single polyphenol compounds were added to the LAL tests, the compounds with caffeoyl and pyrogallol moieties were found to affect the LAL reaction course and recovery rate. Furthermore, none of the compounds had any effects at concentrations of 1 µM. Because the plant extracts contained analogs of various polyphenolic compounds, they were presumed to actually act synergistically. Our findings demonstrated that attention must be paid to the recovery rate and reaction process of LAL tests with samples containing polyphenolic compounds.

Antimicrobial Peptides with Enhanced Salt Resistance and Antiendotoxin Properties.

A strategy was described to design antimicrobial peptides (AMPs) with enhanced salt resistance and antiendotoxin activities by linking two helical AMPs with the Ala-Gly-Pro (AGP) hinge. Among the designed peptides, KR12AGPWR6 demonstrated the best antimicrobial activities even in high salt conditions (NaCl ~300 mM) and possessed the strongest antiendotoxin activities. These activities may be related to hydrophobicity, membrane-permeability, and α-helical content of the peptide. Amino acids of the C-terminal helices were found to affect the peptide-induced permeabilization of LUVs, the α-helicity of the designed peptides under various LUVs, and the LPS aggregation and size alternation. A possible model was proposed to explain the mechanism of LPS neutralization by the designed peptides. These findings could provide a new approach for designing AMPs with enhanced salt resistance and antiendotoxin activities for potential therapeutic applications.

Assessment of interactions of efavirenz solid drug nanoparticles with human immunological and haematological systems.

BACKGROUND: Recent work has developed solid drug nanoparticles (SDNs) of efavirenz that have been demonstrated, preclinically, improved oral bioavailability and the potential to enable up to a 50% dose reduction, and is currently being studied in a healthy volunteer clinical trial. Other SDN formulations are being studied for parenteral administration, either as intramuscular long-acting formulations, or for direct administration intravenously. The interaction of nanoparticles with the immunological and haematological systems can be a major barrier to successful translation but has been understudied for SDN formulations. Here we have conducted a preclinical evaluation of efavirenz SDN to assess their potential interaction with these systems. Platelet aggregation and activation, plasma coagulation, haemolysis, complement activation, T cell functionality and phenotype, monocyte derived macrophage functionality, and NK cell function were assessed in primary healthy volunteer samples treated with either aqueous efavirenz or efavirenz SDN. RESULTS: Efavirenz SDNs were shown not to interfere with any of the systems studied in terms of immunostimulation nor immunosuppression. Although efavirenz aqueous solution was shown to cause significant haemolysis ex vivo, efavirenz SDNs did not. No other interaction with haematological systems was observed. Efavirenz SDNs have been demonstrated to be immunologically and haematologically inert in the utilised assays. CONCLUSIONS: Taken collectively, along with the recent observation that lopinavir SDN formulations did not impact immunological responses, these data indicate that this type of nanoformulation does not elicit immunological consequences seen with other types of nanomaterial. The methodologies presented here provide a framework for pre-emptive preclinical characterisation of nanoparticle safety.

Effect of size and shape on toxicity of zinc oxide (ZnO) nanomaterials in human peripheral blood lymphocytes.

The multi-industrial applications of zinc oxide nanomaterials (ZnO NMs) lead to increasing exposure to humans. Though the ZnO nanoparticles (NPs) toxicity had been evaluated previously, toxicity of other forms of ZnO nanomaterials has not been evaluated. In this study, cytotoxicity and genotoxicity of four different types of ZnO NMs were evaluated using human peripheral blood lymphocytes (HPBL). In addition, the effect of anti-oxidants on ZnO NMs induced toxicity was also evaluated. Our results suggest that, size and shape of the nanomaterials have profound effects on their toxicity. The NPs and nanorods (NRs) possessed higher level of oxidative potential and ROS generation capacity than microparticles (MPs) and microrods (MRs). In contrast, MPs and MRs possessed higher level of lipid peroxidation capacity. The smaller NPs are more genotoxic while larger MPs and MRs were more cytotoxic in nature. Treatment with vitamin C or Quercetin significantly reduces the genotoxicity associated with ZnO NMs. The influence of size and shape in mediating NMs toxicity should be taken into account and the possible supplementation of anti-oxidants might mitigate the toxicity.

Evaluating Intestinal Permeability by Measuring Plasma Endotoxin and Diamine Oxidase in Children with Acute Lymphoblastic Leukemia Treated with High-dose Methotrexate.

BACKGROUND AND AIM: The incidence of acute lymphoblastic leukemia (ALL) is highest in childhood malignant tumor in China. The high-dose methotrexate (HDMTX) treatment is very effective in ALL, and it can improve event-free survival rate. However, while executing the anti-tumor effect, it produces highly toxic effects on rapidly dividing cells which are normal. It seems probable that the HDMTX treatment injures intestinal mucosal barrier. The changes of intestinal mucosal barrier can be evaluated through measuring the level of plasma endotoxin and diamine oxidase (DAO). METHOD: Blood samples were collected from 30 normal children and 30 children with ALL at 1h, 24h, 44h and 68h after HDMTX. The levels of plasma endotoxin and DAO were measured at 1h, 24h, 44h and 68h after HDMTX with spectrophotometry. The levels of endotoxin and DAO were also measured in 4 different courses in 7 children with ALL. RESULTS: The levels of plasma endotoxin and DAO at 1h, 24h, 44h and 68h after HDMTX were higher than in normal children (P<0.01). The levels of plasma endotoxin and DAO at 24h and 44h after HDMTX were both higher than at 1h and 68h (P<0.01). There was no significant difference found in the measured results of plasma endotoxin and DAO at 1h and 68h after HDMTX (P>0.05). There was no significant difference found in the increased levels of endotoxin and DAO at 1h, 24h, 44h and 68h after HDMTX in 4 different courses of 7 children with ALL(P>0.05). CONCLUSION: By measuring the level of plasma endotoxin and DAO in children with ALL and during HDMTX chemotherapy, the results suggest that there is increased intestinal permeability.

Accuracy of Turbidimetric Limulus Amebocyte Lysate Assay for the Recovery of Endotoxin Interacted with Commonly Used Antimicrobial Agents of Endodontic Therapy.

INTRODUCTION: This study was conducted to investigate whether the interaction between the turbidimetric limulus amebocyte lysate (LAL) substrate for endotoxin measurement and the substances/antimicrobial agents used in endodontic therapy can lead to the inhibition/enhancement of endotoxin recovery. METHODS: Ten microliters of a suspension of Escherichia coli endotoxin (O55:B55) was inoculated and kept in contact for 1 hour with different substances categorized as follows: group 1: auxiliary chemical substances: 5.25% and 2.5% sodium hypochlorite solutions, 2% chlorhexidine (CHX) (gel and solution), 1% Natrosol gel (Drogal Chemicals and Pharmaceuticals Ltd, Piracicaba, SP, Brazil), 17% EDTA, 10% citric acid, 3% hydrogen peroxide, 5% sodium thiosulfate, and 0.5% Tween 80 associated with 0.07% soy lecithin (Drogal Chemicals and Pharmaceuticals Ltd) and group 2: intracanal medications: neomycin/polymyxin B/hydrocortisone (Otosporin; Glaxo Wellcome, Rio de Janeiro, RJ, Brazil); calcium hydroxide (Ca[OH]2); Ca(OH)2 + 2% CHX gel; Ca(OH)2 + 2% CHX gel + zinc oxide eugenol; Ca(OH)2 + camphorated paramonochlorophenol (Calen; S.S. White, Rio de Janeiro, RJ, Brazil); triple antibiotic paste; mineral trioxide aggregate (MTA); and iodoform. Positive and negative controls consisted of root canal hemorrhagic exudate and pyrogen-free sterile water, respectively. All samples were diluted up to a 10:4 dilution. Each dilution was individually examined by the turbidimetric LAL assay. Collected data were analyzed through performance characteristics of the LAL assay such as linearity, coefficient of variation percentage, and product positive control (PPC) values. RESULTS: Correlation coefficient (≥0.980) and coefficient of variation percentage (<10%) of the standard curve in triplicate showed the tests' linearity. Spike recovery of auxiliary chemical substances achieved PPC values ranging from 50%-197%, showing no interferences with LAL substrate. Conversely, 3% hydrogen peroxide achieved product inhibition in which endotoxin values were underestimated even after the 10:4 dilutions. Regarding intracanal medicaments, neomycin/polymyxin B/hydrocortisone also inhibited endotoxin detection in all dilutions investigated (PPC values <50%). In contrast, Ca(OH)2 + 2% CHX gel + ZOE as well as triple antibiotic paste led to the enhancement of endotoxin detection in which endotoxin values could not be validated by the turbidimetric kinetic LAL assay (PPC value >200%). CONCLUSIONS: The performance characteristics of the kinetic turbidimetric assay for endotoxin measurement, such as precision and reproducibility, are modulated by the interaction of the LAL substrate with the substances/antimicrobial agents used in endodontic therapy.

Single session of Nd:YAG laser intracanal irradiation neutralizes endotoxin in dental root dentin.

Endotoxins released in the dental root by Gram-negative microorganisms can be neutralized by calcium hydroxide, when this medication is applied inside the root canal for at least seven days. However, several clinical situations demand faster root canal decontamination. Thus, for faster endotoxin neutralization, endodontists are seeking additional treatments. The in vitro study tested whether or not intracanal Nd:YAG laser irradiation would be able to neutralize endotoxin within the human dental root canal in a single session. Twenty-four human teeth with one root were mounted between two chambers. After conventional endodontic treatment, root canals were contaminated with Escherichia coli endotoxin. Then they were irradiated or not (controls) in contact mode with an Nd:YAG laser (1.5 W, 15 Hz, 100 mJ and pulse fluency of 124 J/cm2). The endotoxin activity was measured using the limulus lysate technique and data were statistically compared (p≤0.05). The concentration of active endotoxin measured in the negative control group was significantly lower than that of the positive control group (p=0.04). The concentrations of endotoxin in both irradiated groups were significantly lower than that of the positive control group (p=0.027) and similar to that of negative control group (p=0.20). A single session of intracanal Nd:YAG laser irradiation is able to neutralize endotoxin in the dental root tissues.

Efficacy of endodontic treatment for endotoxin reduction in primarily infected root canals and evaluation of cytotoxic effects.

INTRODUCTION: Endotoxins are one of the etiologic agents involved in the pathogenesis of apical periodontitis. The objectives of this clinical study were to investigate the effects of endodontic treatment by using different irrigants on endotoxins in root canals with pulp necrosis and apical periodontitis and to evaluate the cytotoxic effects. METHODS: Thirty-six root canals were selected. Samples were collected before (S1) and after instrumentation (S2). The root canals were divided into 3 groups (n = 12) according to the irrigant combination used: CLX + LW, 2% chlorhexidine gel + calcium hydroxide (0.14%, limewater); CLX + PmB, chlorhexidine + polymyxin B; CLX (control), chlorhexidine + saline. The third sampling (S3) was performed after ethylenediaminetetraacetic acid and S4 after intracanal medication (CLX + calcium hydroxide for 14 days). Endotoxins were quantified by the chromogenic Limulus amebocyte lysate assay, and cytotoxic effects were evaluated by the production of cytokines (interleukin-1ß, tumor necrosis factor α) in macrophages (RAW 264.7) stimulated with the root canal content. RESULTS: Endotoxins were detected in all root canals before instrumentation (S1). Group CLX + LW presented the greatest endotoxin reduction after instrumentation (99.18%), which was similar to group CLX + PmB (96.42%, P > .05) and different from group CLX (90.78%, P < .05). The intracanal medication promoted important endotoxin neutralization, with a reduction of 99.2% to 100%. The root canal content induced a higher production of tumor necrosis factor α and interleukin-1ß in S1 samples compared with samples obtained after treatment. CONCLUSIONS: The combination of CLX and limewater as irrigant was the most effective in reducing endotoxins in root canals, and intracanal medication was important to neutralize the cytotoxic effects.

Effectiveness of castor oil extract on Escherichia coli and its endotoxins in root canals.

This in vitro study sought to evaluate the effectiveness of castor oil extract used as an irrigating solution on Escherichia coli and its endotoxins in root canals. Sixty single-rooted teeth were prepared (using castor oil extract as irrigating solution) and divided into five groups (n = 12): Group 1 samples were treated with calcium hydroxide (Ca(OH)2), Group 2 samples were treated with polymyxin B, Group 3 samples were treated with Ca(OH)2 and 2% chlorhexidine gel (CHX), and Group 4 samples were treated with castor oil extract. A control group used physiological saline solution as an irrigant. Canal content samples were collected at four different times: immediately after instrumentation, seven days after instrumentation, after 14 days of intracanal medication, and seven days after removal of intracanal medication. A plating method was used to assess antimicrobial activity and the quantification of endotoxins was evaluated by the chromogenic Limulus lysate assay. Data were submitted to ANOVA and a Dunn test (a = 5%). Irrigation with castor oil extract decreased E. coli counts but had no effect on the level of endotoxins. Samples taken seven days after removal of medication revealed a significant reduction in endotoxin levels in Groups 3 and 4. Compared to the saline solution irrigation, castor oil extract decreased microorganism counts in root canals immediately after canal preparation. None of the medications used completely eliminated endotoxins in the root canal.

Characterization of blood beta-1,3-glucan and anti-beta-glucan antibody in hemodialysis patients using culinary-medicinal Royal Sun Agaricus, Agaricus brasiliensis S. Wasser et al. (Agaricomycetideae).

Beta-glucan is a major component of fungal cell walls and shows various immunopharmacological activities including antitumor activity. Previously, we detected anti-beta-glucan antibody in human sera. Anti-beta-glucan antibody participates in the immune response to fungal cell wall beta-glucan. Patients on dialysis are at high risk of infection including fungal infections. We examined the plasma beta-glucan level and the titer of anti-beta-glucan antibody in dialysis patients. We measured plasma beta-1,3-glucan concentrations with the limulus G test and anti-beta-glucan antibody titers by ELISA with Candida beta-glucan-coated plates. We also examined the influence of the period of dialysis and the kind of dialysis membrane. The patients were positive for beta-1,3-glucan in their plasma. The anti-beta-glucan antibody titer was lower in the dialysis patients than in healthy volunteers. Long-term dialysis patients showed lower anti-beta-glucan antibody titers than short-term dialysis patients. No significant difference was found between the kinds of dialysis membrane. The titer of anti-beta-glucan antibody as recognition molecule of beta-glucan was low in dialysis patients compared with healthy volunteers. This is likely to be one factor explaining the sensitivity to infection of the dialysis patients. An appropriate application of culinary-medicinal mushroom such as Agaricus brasiliensis has potential for the prevention of fungal infection in dialysis patients.

Functional characterization of lipopolysaccharide derived from symbiotic bacteria in rice as a macrophage-activating substance.

BACKGROUND AND AIM: Lipopolysaccharide derived from a symbiotic bacterium in wheat (Pantoea agglomerans, LPSp) has shown multiple positive effects, such as prophylactic, antiallergic and antitumour effects, without serious side-effects. LPSp has differential biological activities in comparison to other LPS, such as those from Escherichia coli (LPSe). The only difference between LPSp and LPSe is in the O-antigen polysaccharide structure (O-PS). This led us to the hypothesis that the O-PS structure would seem to participate in biological activities. Thus, the characterization of properties of O-PS in LPS is of the utmost importance for understanding cell activation in the maintenance of homeostasis. However, little is known about the correlation between the O-PS structure of LPS and its biological activities. In this study, we extracted LPS derived from a symbiotic bacterium in rice (strain A46, related species of Pantoea), which has a long history of use in foods, and investigated its putative structures and functions. MATERIALS AND METHODS: LPS derived from strain A46 was prepared using a hot phenol extraction method. The properties of LPS-A46 were analysed by thin-layer chromatography, Tricine SDS-PAGE and Western blotting. The function of LPS-A46 was analyzed by quantative real-time PCR and flow cytometry using THP-1 cells and Peripheral blood mononuclear cell (PBMC) derived macrophages. RESULTS: In Tricine SDS-PAGE, high molecular mass LPS-A46 had a molecular mass lower than that of LPSp. In Western blotting, LPS-A46 reacted with lipid A antibody but did not react with an O-PS antibody of LPSp. In comparison to other LPS, LPS-A46 induced a differential cytokine gene expression profile in THP-1 cells and PBMC-derived macrophages. CONCLUSION: The present study suggests that LPS derived from symbiotic bacterium in rice is a bioactive functional LPS which may have different functional activities compared to other types of LPS.

Effect of Escherichia coli endotoxin on Archachatina marginata hemolymph coagulation system.

CONTEXT: Archachatina marginata Swainson (Achatinidae) is found in Nigeria, West Africa. Its hemolymph is applied as a disinfectant to blades and fresh cuts of circumcision in Yorubaland. The hemolymph is also used in traditional medicine practice. Investigation into its anti-endotoxin response is being studied for the first time. OBJECTIVE: This study determined whether endotoxin causes measurable and concentration-dependent protein coagulation in the separate hemolymph fractions and in hemocyte lysate (HL)/plasma mixtures. MATERIALS AND METHODS: Endotoxin was prepared by inoculating 5% w/v dextrose with locally isolated Escherichia coli cells and incubated for 48 h before sterilization. Pyrogenicity was determined by rabbit test method and use the of LAL kit. Hemolymph fractions were exposed to endotoxin while controls were exposed to endotoxin-free water (0.025 EU/ml). HL/plasma (1:1 v/v) was exposed to varied endotoxin concentrations. RESULTS: Data indicated significantly higher protein coagulates induced by endotoxin in all the hemolymph fractions (P < 0.05). Maximum protein coagulation in mixture of HL/plasma 1:1 was recorded. Exposure of HL/plasma at optimal ratio to varied endotoxin caused linear protein coagulation up to 1.0 EU/ml, beyond which it dropped significantly and unresponsive to further increase in endotoxin doses. DISCUSSION AND CONCLUSION: There was endotoxin-induced protein coagulation, which is endotoxin concentration-dependent. The optimal coagulation observed for 1:1 HL/plasma mixture suggests stronger interaction between the hemocytes and the plasma in response to endotoxin. There are LPS-binding proteins in the plasma and hemocytes of A. marginata. This finding may be employed in detection and quantification of endotoxin in future.

[Content detection of bacterial endotoxin in two kinds of injection by gelatin technique].

OBJECTIVE: To detect content of bacterial endotoxin in Yuxingcao and Qingkailing injections by specific and nonspecific tachypleus amebocyte lysate technique for in order to investigate the feasibility of specific tachypleus amebocyte lysate technique for detecting bacterial endotoxin in traditional Chinese drug injections. METHOD: Different batches of Yuxingcao and Qingkailing injections were detected by specific and nonspecific tachypleus amebocyte lysate kits. RESULT: Yuxingcao injection could be detected by specific and nonspecific tachypleus amebocyte lysate technique, Whereas Qingkailing injection could be detected only by specific tachypleus amebocyte lysate. CONCLUSION: Using specific tachypleus amebocyte lysate as a substitute for nonspecific tachypleus amebocyte lysate is an effective method for detecting content of bacterial endotoxin in Qingkailing injection.

Xanthohumol feeding does not impair organ function and homoeostasis in mice.

Xanthohumol, the major prenylated chalcone found in hops, is known to exert several beneficial effects but only few studies evaluated the safety profile of this natural compound with in part discrepant results. Here, we fed female BALB/c mice with a standard diet supplemented with xanthohumol for 3 weeks, and thus, achieved a daily dose of approximately 1000 mg xanthohumol/kg body weight. There were no significant differences in body weight or food intake between mice on standard diet and animals receiving the same diet supplemented with xanthohumol. Histopathological examination of liver, kidney, colon, lung, heart, spleen and thymus revealed no signs of xanthohumol-toxicity, and biochemical serum analysis confirmed normal organ function. Further, xanthohumol treatment did not affect hepatic glycogen content CYP2E1 and CYP1A2 expression levels, but CYP3A11 mRNA was approximately 30% reduced. Expression of several genes indicative of early hepatic inflammation and fibrosis, a hallmark of chronic liver injury, did not differ between xanthohumol treated and control mice. In summary, these results indicate that oral administration of xanthohumol exhibits no adverse effects on major organ function and homoeostasis in mice. Particularly, hepatotoxic effects could be ruled out confirming a good safety profile of xanthohumol as prerequisite for further studies in humans.

Content detection of bacterial endotoxin in two kinds of injection by gelatin technique / 中国中药杂志

<p><b>OBJECTIVE</b>To detect content of bacterial endotoxin in Yuxingcao and Qingkailing injections by specific and nonspecific tachypleus amebocyte lysate technique for in order to investigate the feasibility of specific tachypleus amebocyte lysate technique for detecting bacterial endotoxin in traditional Chinese drug injections.</p><p><b>METHOD</b>Different batches of Yuxingcao and Qingkailing injections were detected by specific and nonspecific tachypleus amebocyte lysate kits.</p><p><b>RESULT</b>Yuxingcao injection could be detected by specific and nonspecific tachypleus amebocyte lysate technique, Whereas Qingkailing injection could be detected only by specific tachypleus amebocyte lysate.</p><p><b>CONCLUSION</b>Using specific tachypleus amebocyte lysate as a substitute for nonspecific tachypleus amebocyte lysate is an effective method for detecting content of bacterial endotoxin in Qingkailing injection.</p>

Extraction of the anti-sepsis component from Terminaliachebula Retz and evaluation of its biological activities.

Many clinical experiments and studies have demonstrated that traditional Chinese medicines possess the capacity for being used in anti-sepsis. In this paper, we screened 78 herbs based on biosensor technology by targeting of lipid A. Terminaliachebula Retz was found to possess the highest capability of binding lipid A. With CER (cation-exchange resin) and HPLC, we obtained three active components extracted from Terminaliachebula Retz, and named them TCR1, TCR2 and TCR3 respectively. These three components were evaluated with the biosensor, and it was found that the TCR3 was the most capable candidate to bind lipid A. We also studied the biological activities of TCR3 against sepsis in vitro and in vivo. in vitro, TCR3 could significantly inhibit LPS (lipopolysaccharide)-induced LAL (Limulus amoebocyte lysate)) from agglutination and decrease TNFalpha (tumour necrosis factor alpha) release from RAW264.7 cells induced by LPS in a dose-dependent manner. in vivo, TCR3 could significantly protect mice against a lethal challenge with LPS and heat-killed Escherichia coli 35218 in a dose-dependent manner. These results demonstrate that Terminaliachebula Retz is an important herb to neutralize LPS and it has the potential to serve as a treatment for sepsis.

A multicenter comparison study between the Endosafe PTS rapid-release testing system and traditional methods for detecting endotoxin in cell-therapy products.

BACKGROUND: Rapid-release testing reduces the waiting period for administration of time-sensitive cell-therapy products. Current assay systems are labor intensive and time consuming. The Endosafe portable test system (PTS) is a chromogenic Limulus amebocyte lysate (LAL) portable endotoxin detection system that provides quantitative results in approximately 15 min. To evaluate Endosafe performance with cell-therapy products, side-by-side testing of traditional LAL systems and the Endosafe system was conducted at the Production Assistance for Cellular Therapies (PACT) facilities and the National Institutes of Health's Department of Transfusion Medicine, USA. METHODS: Charles River Laboratories provided each center with a PTS reader and two commercially prepared lyophilized reference standard endotoxin (RSE) vials. All samples tested with the Endosafe system used 0.05-5.0 endotoxin unit/mL (EU/mL) sensitivity cartridges provided by Charles River. Each vial was reconstituted with LAL water and tested in triplicate using the Endosafe and in-house LAL methods. Subsequently, each center tested the endotoxin content of standard dilutions of cell-therapy products, thus creating paired test results for each sample. Additionally, fabricated endotoxin-positive samples containing varying concentrations of endotoxin were prepared and shipped to all centers to perform blinded testing. RESULTS: Valid paired results, based on each center's LAL method and the Endosafe system criteria, were analyzed. Endotoxin detection between paired results was equivalent in most cases. DISCUSSION: The Endosafe system provided reliable results with products typically produced in cell-therapy manufacturing facilities, and would be an appropriate test on which to base the release of time-sensitive cell-therapy products.

Early effects triggered by Larrea divaricata Cav. on murine macrophages at apoptotic concentrations.

Decoction and infusion of Larrea divaricata were tested at apoptotic concentrations (1 and 4 mg/ml) on peritoneal murine macrophages. Consistent changes were observed after incubation with 4 mg/ml decoction. Phagocytosis of zymosan, lysosomal enzyme activity, nitric oxide production, TNF-alpha release, and expression of CD14, TLR4, and CR3 increased significantly. Decoction at 1 and 4 mg/ml increased the binding of LPS-FITC. Apoptosis triggered by L. divaricata decoction is consequence of cell activation. The effects are independent of nordihydroguaiaretic acid. This "activation and death" could be the mechanism of L. divaricata to exert the antituberculosis effect known in folk medicine.

[Study of lipopolysaccharide antagonizing effect of DPR-2 in vitro].

OBJECTIVE: To investigate the lipopolysaccharide (LPS) antagonizing biological activity of densefruit pattany root-bark extract (DPR-2) in vitro. METHODS: The effect of DPR-2 in neutralizing LPS (0.1 microg/L) was detected by kinetic turbidimetric limulus test. The effect of different concentrations of DPR-2 (0,8.0,16.0,32.0,64.0 mg/L) on binding of FITC-conjugated LPS (FITC-LPS,100.0 microg/L) to murine RAW264.7 cells was analyzed with laser scanning confocal microscopy. The expression of TNF-alpha and IL-6 mRNA in RAW264.7 cells after exposure to LPS (100.0 microg/L) were determined by real-time RT-PCR. RESULTS: DPR-2 could neutralize LPS (P < 0.05 or P < 0.01), and inhibit the binding of FITC-LPS to RAW264.7 cells in a dose-dependent manner when the concentration of DPR-2 was above 16.0 mg/L. Furthermore, DPR-2 could markedly inhibit the expression of TNF-alpha and IL-6 mRNA in LPS-stimulated murine RAW264.7 cells. CONCLUSION: DPR-2 exhibit an anti-LPS effect in vitro, which may be related to its capacity to neutralize LPS and inhibit binding of LPS for its receptors.

Purification and protective efficacy of monomeric and modified Yersinia pestis capsular F1-V antigen fusion proteins for vaccination against plague.

The F1-V vaccine antigen, protective against Yersinia pestis, exhibits a strong tendency to multimerize that affects larger-scale manufacture and characterization. In this work, the sole F1-V cysteine was replaced with serine by site-directed mutagenesis for characterization of F1-V non-covalent multimer interactions and protective potency without participation by disulfide-linkages. F1-V and F1-V(C424S) proteins were overexpressed in Escherichia coli, recovered using mechanical lysis/pH-modulation and purified from urea-solubilized soft inclusion bodies, using successive ion-exchange, ceramic hydroxyapatite, and size-exclusion chromatography. This purification method resulted in up to 2mg/g of cell paste of 95% pure, mono-disperse protein having < or =0.5 endotoxin units per mg by a kinetic chromogenic limulus amoebocyte lysate reactivity assay. Both F1-V and F1-V(C424S) were monomeric at pH 10.0 and progressively self-associated as pH conditions decreased to pH 6.0. Solution additives were screened for their ability to inhibit F1-V self-association at pH 6.5. An L-arginine buffer provided the greatest stabilizing effect. Conversion to >500-kDa multimers occurred between pH 6.0 and 5.0. Conditions for efficient F1-V adsorption to the cGMP-compatible alhydrogel adjuvant were optimized. Side-by-side evaluation for protective potency against subcutaneous plague infection in mice was conducted for F1-V(C424S) monomer; cysteine-capped F1-V monomer; cysteine-capped F1-V multimer; and a F1-V standard reported previously. After a two-dose vaccination with 2 x 20 microg of F1-V, respectively, 100%, 80%, 80%, and 70% of injected mice survived a subcutaneous lethal plague challenge with 10(8) LD(50)Y. pestis CO92. Thus, vaccination with F1-V monomer and multimeric forms resulted in significant, and essentially equivalent, protection.