Chromogenic Limulus Amoebocyte Lysate Research Articles

Clinical efficacy of endodontic protocols on reducing cultivable bacteria and endotoxin in infected root canal in patients submitted to head and neck radiotherapy: a randomised clinical trial.

Assess the efficacy of biomechanical preparation using a reciprocating system followed by final irrigation protocols, then intracanal medication, on reducing endotoxins and cultivable bacteria of infected teeth in irradiated patients. Twenty-two infected single-rooted canals in patients submitted to head and neck radiotherapy were prepared by reciprocating motion and 2.5% NaOCl. Patients were randomly divided into two groups of 11 patients before the final irrigation protocol: apical positive pressure (APP) or passive ultrasonic activation (PUA). Both groups were treated in two sessions, using Ca(OH)2 as intracanal medication for 14 days. Root canal content sampling was performed after canal access (S1), after biomechanical preparation plus the irrigation protocol (S2), and after intracanal medication (S3). Chromogenic limulus amoebocyte lysate assay measured endotoxin levels (EU/mL), and bacterial load was determined by culture techniques (CFU/mL). Treatment protocols reduced bacterial counts after S2 in both groups (p = 0.01). S3 differed from S1 (p = 0.01), but not from S2 (p = 0.4). Endotoxin levels were reduced in both groups after S2 (P = 0.03) and were lower in S3 than in S2, with significant differences in the APP group (p = 0.03). Biomechanical preparation using a reciprocating system and 2.5% NaOCl in irradiated teeth, followed by the irrigation protocol (APP or PUA), demonstrated efficacy in reducing endodontic contaminants. Ca(OH)2 as intracanal medication should be performed in irradiated patients with infected root canals. This clinical study demonstrated that endodontic treatment in irradiated patients is efficacious at reducing bacterial load and endotoxin levels.

Indoor Airborne Microbiome and Endotoxin: Meteorological Events and Occupant Characteristics Are Important Determinants.

Airborne bacteria and endotoxin may affect asthma and allergies. However, there is limited understanding of the environmental determinants that influence them. This study investigated the airborne microbiomes in the homes of 1038 participants from five cities in Northern Europe: Aarhus, Bergen, Reykjavik, Tartu, and Uppsala. Airborne dust particles were sampled with electrostatic dust fall collectors (EDCs) from the participants' bedrooms. The dust washed from the EDCs' clothes was used to extract DNA and endotoxin. The DNA extracts were used for quantitative polymerase chain (qPCR) measurement and 16S rRNA gene sequencing, while endotoxin was measured using the kinetic chromogenic limulus amoebocyte lysate (LAL) assay. The results showed that households in Tartu and Aarhus had a higher bacterial load and diversity than those in Bergen and Reykjavik, possibly due to elevated concentrations of outdoor bacterial taxa associated with low precipitation and high wind speeds. Bergen-Tartu had the highest difference (ANOSIM R = 0.203) in β diversity. Multivariate regression models showed that α diversity indices and bacterial and endotoxin loads were positively associated with the occupants' age, number of occupants, cleaning frequency, presence of dogs, and age of the house. Further studies are needed to understand how meteorological factors influence the indoor bacterial community in light of climate change.

Exposure to dust, endotoxins and microbes in Finnish vegetable greenhouses.

In Finland and other northern countries, vegetable greenhouse workers are exposed to airborne dust and microbes all year round. The aim of the study was to assess respiratory exposure to dust, endotoxins and microbes, and to identify the risk phases of respiratory and skin exposure to promote safe working methods. Work in greenhouses was observed and recorded in a structured form. 23 personal samples and eight stationary samples of dust, endotoxins and viable microbes were collected from eight tomato or cucumber greenhouses in Finland. Dust samples were analysed gravimetrically. Endotoxins were analysed on filters by chromogenic Limulus amoebocyte lysate assay. Microbes were collected on filters, incubated and counted in three different culture media. Eight additional stationary samples of microbes were collected on a six-phase impactor. The greenhouses were generally clean and well organised, but skin protection was deficient. The median of personal dust exposure was 0.24 mg/m3: 0.21 mg/m3 in the tomatohouses and 0.62 mg/m3 in the cucumber greenhouses. The mean level of bacteria in the tomato houses was 2.7 × 103 and in the cucumber houses 1.6×104. Those of fungi were 3.6 × 105 and 1.7 × 105 on Hagem and 8.1 × 105 and 1.9 × 105 on DG-18, respectively. The microbes made up about 1/10 of these values in the stationary samples. Very low concentrations of endotoxins were found in 5/23 air samples. The clean appearance of the greenhouses was reflected in the low to moderate levels of dust. Risk phases of high exposure, such as lifting leaf debris, were identified. The microbe species were typical of this climate, and their levels were comparable to other greenhouse studies, but lower than in farming. Prevention among the foreign workforce is challenging due to the lack of a common language.

Comparison of LAL and rFC Assays-Participation in a Proficiency Test Program between 2014 and 2019.

Endotoxin (lipopolysaccharide) testing of drugs is routinely required in pharmaceutical industries. Suitable compendial assays are defined by national pharmacopoeias. At this time, Limulus Amoebocyte Lysate (LAL) assays are the gold standard. LAL is used in vitro for specific detection of endotoxin based on endotoxin-activated Factor C-mediated clotting cascade. However, alternative mediated pathways (e.g., Factor G), impurities, and further factors may influence test results. Some of these influencing factors are eliminated by recombinant Factor C (rFC) test, which represents a promising alternative. rFC not only enables highly specific endotoxin testing, as interfering Horseshoe Crab blood components are eliminated, but also offers ethical and ecological advantages compared to classical LAL assays. However, the question remains whether rFC-based tests are robust test systems, equivalent or superior to LAL and suitable for routine bacterial endotoxin testing. Pharmaceutical test users have validated the test successfully for their specific products, but no long-term studies have been published that combine testing of unknown samples, inter-laboratory, -operator, and -lot changes. Thus, it was of great interest to investigate rFC test performance in a routine setting within a proficiency test program set-up. Over a period of six years comparative endotoxin testing was conducted with one kinetic chromogenic LAL assay and two rFC-based assays. Results of this study demonstrate that both rFC-based assays were comparable to LAL. All results met acceptance criteria defined by compendial bacterial endotoxin testing. RFC-based methods generated results with even better endotoxin recovery rates compared to LAL. Therefore, rFC-based tests were found to represent reliable methods, as equivalent or even superior to LAL assays and suitable for routine bacterial endotoxin testing.

Occurrence of bacteria and endotoxins in fermented foods and beverages from Nigeria and South Africa

In Africa, fermented foods and beverages play significant roles in contributing to food security. Endotoxins are ubiquitous heat stable lipopolysaccharide (LPS) complexes situated in the outer cell membranes of Gram-negative bacteria. This study evaluated the microbiological quality of fermented foods (ogiri, ugba, iru, ogi and ogi baba) and beverages (mahewu and umqombothi) from selected Nigerian and South African markets. The bacterial diversity of the fermented foods was also investigated and the identity of the isolates confirmed by biochemical and molecular methods. Isolate grouping was established through hierarchal clustering and the samples were further investigated for endotoxin production with the chromogenic Limulus Amoebocyte Lysate assay. The total aerobic count of the samples ranged from 5.7 to 10.8 Log CFU/g. Fourteen bacteria genera were detected with most of the isolates being members of the Enterobacteriaceae family. Sphingomonas paucimobilis and Escherichia coli were the dominant Gram-negative bacterial species detected. There were considerable variations in the concentrations of endotoxins produced and the lowest endotoxin concentration was found in ogi (4.3 × 10 EU/g) and the highest in iru (5.5 × 104 EU/g) while, 44% of umqombothi samples had endotoxins. Ogi baba samples had better microbial quality than other samples due to its reduced bacterial load and endotoxin levels. There was a strong positive (r = 0.714, r = 0.996) and significant (p < 0.01) correlation between the endotoxin levels and bacterial loads of the samples. Some previously unreported species of bacteria found in the fermented foods included Aeromonas haemolyticus and Rhizobium radiobacter. This is the first comprehensive report on endotoxins in fermented foods and beverages in Africa. Furthermore, the occurrence of pathogenic bacteria and toxins in the foods and beverages is of serious concern that calls for immediate action.

Reduction of Endotoxin from Human Root Canals by Calcium Hydroxide Nanoparticles

This study was aimed to evaluate the reduction of endotoxin using the passive ultrasonic irrigation (PUI) of calcium hydroxide nanoparticles (CHNPs) compared with the PUI of calcium hydroxide (CH) and the CH medication. Forty-seven single-root human premolars were used. The root canals were enlarged and sterilized. Escherichia coli endotoxin was inoculated into the root canals. Thirty-six root canals were assigned into three experimental groups: CHNPs irrigation, CH irrigation, and CH medication. For the irrigation groups, the PUI was activated for three min. For the CH medication, the CH was filled into each canal and incubated at 37°C for seven days. The reduction of endotoxin was evaluated by quantitative chromogenic limulus amoebocyte lysate assay. Data was analysed by Kruskal-Wallis and pairwise comparison tests, with a level of significance set at P < 0.05. The CHNPs irrigation was more effective in against endotoxin than the CH irrigation (P < 0.05). No significant difference was found between the CHNPs irrigation and the CH medication (P > 0.05). The irrigation of CHNPs could eliminate most of the endotoxin from the root canals and approximately from 300 μm of root dentin. CHNPs might be potential as an adjunctive irrigation for endotoxin removal.

The relationship of endotoxaemia to peripheral and central nervous system inflammatory responses in Human African Trypanosomiasis.

Endotoxaemia has been described in cases of Human African trypanosomiasis (HAT), but it is unclear if this phenomenon influences inflammatory pathology either in the periphery or central nervous system (CNS). We studied endotoxin concentrations in the plasma and cerebrospinal fluid (CSF) of Trypanosoma brucei rhodesiense patients using the chromogenic Limulus Amoebocyte lysate assay. The relationship of endotoxin concentration to the presentation of gross signs of inflammation and the inflammatory/counter-inflammatory cytokine profile of the relevant compartments were analysed. We demonstrate that HAT patients exhibit parasitaemia-independent plasma endotoxaemia, and that this is associated with splenomegaly and lymphadenopathy. Endotoxin concentrations normalize rapidly after treatment. There was no evidence of endotoxin release in the CNS. A rapid normalization of endotoxin levels after treatment and lack of association with parasitaemia suggest that gut leakage is the main source of endotoxin in the circulation. Low CSF endotoxin concentrations and a lack of any association with neuroinflammatory markers or neurological sequelae suggest that endotoxin does not play a role in the pathogenesis of the disease in the CNS.

A comparative study of blood endotoxin detection in haemodialysis patients.

BackgroundEndotoxemia is commonly reported in patients receiving haemodialysis and implicated in the pathogenesis of systemic inflammation. The Limulus Amoebocyte Lysate (LAL) assay is the most commonly used blood endotoxin detection assay. Two kinetic variations of the assay are commercially available – the turbidimetric and chromogenic assay, it is unknown which assay is superior for endotoxin detection in uremic patients. Selection of the optimum LAL technique for endotoxin detection in haemodialysis patients is important to further understanding of the sequela of endotoxemia and development of endotoxin-lowering strategies in this population.MethodA turbidimetric and chromogenic LAL assay from the same manufacturer were directly compared. We investigated the ability of both LAL assays to detect endotoxin in uremic plasma. Plasma samples from haemodialysis patients and healthy controls were spiked with endotoxin and percentage spike recovery for the chromogenic and turbidimetric assay was determined. Assay accuracy and precision were compared between both LAL assays.ResultsThe turbidimetric assay had greater accuracy than the chromogenic assay. Spike recovery was 113.8 % vs. 53.8 % for the turbidimetric and chromogenic assay respectively. Assay bias was higher in the chromogenic assay (−0.384EU/mL vs. 0.011EU/mL). The turbidimetric assay demonstrated greater precision compared to the chromogenic assay. Coefficient of variation ranged from 4.5 to 24.1 % for the turbidimetric assay and 25.8–26.5 % for the chromogenic assay.ConclusionThe study findings suggest that the kinetic turbidimetric LAL assay has greater accuracy and precision than the chromogenic assay and is the optimum LAL technique for endotoxin detection in haemodialysis patients, though these findings should be verified using LAL reagents from other sources.

Optimising the use of commercial LAL assays for the analysis of endotoxin contamination in metal colloids and metal oxide nanoparticles

Engineered nanoparticles (NP) are generally contaminated by bacterial endotoxin, a ubiquitous bacterial molecule with significant toxic and inflammatory effects. The presence of endotoxin, if not recognised, can be responsible for many of the in vitro and in vivo effects attributed to NPs. The Limulus Amoebocyte Lysate (LAL) assay, the test requested by regulatory authorities for assessing endotoxin contamination in products for human use, is not immediately applicable for testing endotoxin in NP preparations, mainly due to the possible interference of NPs with the assay readouts and components. In this study, we have compared different commercially available LAL assays for detecting endotoxin in gold, silver and iron oxide NPs. Different NP chemistry, concentrations and surface coatings could differently interfere with the LAL assays’ results. After accurate testing of the possible interaction/interference of NPs with the various assay components, the modified chromogenic LAL assay proved the most suitable assay for measuring endotoxin in NP samples, provided the appropriate controls are performed. Thus, endotoxin determination can be performed in NP preparation with commercial LAL assays only after assay validation, i.e. once possible interference of NPs with the assay components and readouts has been excluded.

Choice of Method for Endotoxin Detection Depends on Nanoformulation

Many nanoparticles interfere with traditional tests to quantify endotoxin. The aim of this study was to compare the performance of limulus amoebocyte lysate (LAL) formats on clinical-grade nanoformulations, to determine whether there were disparate results among formats and to test the applicability of an alternative bioassay (the macrophage activation test [MAT]) for resolving discrepancies, if observed. Clinical-grade nanoformulations were tested using turbidimetric, gel-clot and chromogenic LAL. Formulations that cause a discrepancy among LAL tests were also tested by the MAT. The gel-clot LAL method cannot be relied upon to resolve discrepancies among LAL tests for certain nanoformulations. No one LAL format was shown to be optimal for all the tested clinical-grade nanoformulations. The tested alternative bioassay (the MAT) was useful for verifying LAL findings, but only for those nanoformulations not carrying/including cytotoxic drugs.

1162 PROSPECTIVE STUDY ASSESSING THE PRESENCE OF ENDOTOXAEMIA IN PATIENTS UNDERGOING TRANSRECTAL PROSTATE BIOPSY

You have accessJournal of UrologyInfections/Inflammation of the Genitourinary Tract: Prostate & Genitalia1 Apr 20131162 PROSPECTIVE STUDY ASSESSING THE PRESENCE OF ENDOTOXAEMIA IN PATIENTS UNDERGOING TRANSRECTAL PROSTATE BIOPSY Sharon Sheehan, John Philpott-Howard, Wei Wang, Hemant Nemade, and Peter Thompson Sharon SheehanSharon Sheehan London, United Kingdom More articles by this author , John Philpott-HowardJohn Philpott-Howard London, United Kingdom More articles by this author , Wei WangWei Wang Beijing, China, People's Republic of More articles by this author , Hemant NemadeHemant Nemade London, United Kingdom More articles by this author , and Peter ThompsonPeter Thompson London, United Kingdom More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.799AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Despite antibiotic prophylaxis, the risk of sepsis with transrectal prostate biopsy (TRPB) is well recognised. This was a prospective bacteriological study of consecutive patients undergoing TRPB. Uniquely we examined in all patients the prevalence of endotoxaemia following biopsy and its association with the results of bacteriological cultures. METHODS 67 consecutive patients were enrolled in the study from January to July 2011. Each patient received standard prophylaxis with ciprofloxacin and metronidazole. Blood cultures and serum for endotoxin assay were collected at 5min, 60 min and 24 hours post biopsy. Prostate needle washings were also cultured. The presence of endotoxin was determined utilising the chromogenic Limulus Amoebocyte Lysate (LAL) assay. RESULTS 61/67 patients (91.0%) had positive cultures from prostate biopsy needle washings. 51/67 (76.1%) patients had Gram positive bacteria cultured; 23/67 (34.3%) of patients had Gram negative bacteria cultured. 6/67 patients (9.0%) had positive blood cultures. All of these organisms cultured were Gram positive. A total of 66 samples were received for endotoxin at 5 min, 60 samples at 60min and 60 samples at 24 h post biopsy. The lower limit of detection of the assay was 0.005 Endotoxin Units (EU)/mL and any value under this was considered undetectable; values greater than 1.000 EU/mL were considered highly significant. Endotoxin levels at the various time points were as follows: At five minutes 62/66 patients (94.0%) had detectable endotoxin, mean 2.152 EU/mL (SD +/− 4.011). Of these 21/66 (31.8%) had >1.000 EU/mL. At 60 minutes 53/60 patients (88.3%) had detectable endotoxin, mean 2.041 EU/mL (SD +/− 4.293). Of these 16/60 (26.6%) had >1.000 EU/mL. At 24 h 55/60 patients (91.6%) had detectable endotoxin, mean 2.583 EU/mL (SD +/− 4.392). Of these 24/60 (40.0%) had >1.000 EU/mL. None of the enrolled patients presented with signs or symptoms suggestive of infection after biopsy. CONCLUSIONS This study provides evidence for translocation of potentially harmful gut endotoxin during TRPB, with high levels of endotoxin detectable in approximately one-third of patients. In conjunction with the bacteraemias, this serves to highlight the potential risk of infection associated with this procedure. Clinicians may not appreciate the risk of translocation of endotoxin; other approaches to biopsy such as the transperineal route may reduce the potential for endotoxemia and infective complications. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e474-e475 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Sharon Sheehan London, United Kingdom More articles by this author John Philpott-Howard London, United Kingdom More articles by this author Wei Wang Beijing, China, People's Republic of More articles by this author Hemant Nemade London, United Kingdom More articles by this author Peter Thompson London, United Kingdom More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Environmental and occupational respiratory diseases – 1042. Influence of environmental endotoxin and allergen levels on prevalence of asthma and allergen sensitizations in urban and rural children in Guandong, China

Methods Three hundred and seventeen children aged 13-14 years (Guangzhou 188, Conghua 129) with self-reported respiratory symptoms and 537 healthy children were selected from 854 school-children according to their response to ISAAC standardized questionnaire. These children attended the school infirmary again to complete a detailed questionnaire and medical examinations including skin prick test (SPT) to 8 common aeroallergens, lung function test, histamine bronchial provocation test and peripheral blood eosinophil counts. House dust samples were collected from living room and mattresses of 156 of these children with the agreement of their parents (Guangzhou 76, Conghua 80). The content of endotoxin in house dust was quantified using an endpoint chromogenic kinetic limulus amoebocyte lysate test. Associations between levels of house dust allergens and endotoxin and the provocation dose producing a 20% fall in forced expiratory volume in one second (PD20-FEV1) and allergen skin reactions were analysed by logistic regression. Results The prevalence of wheeze symptom, asthma, nasal symptoms and allergic rhinitis in the children of Guangzhou were significantly higher (28.6%, 27.7%, 66%, 46.4%, respectively) than the children of Conghua (5%, 2.5%, 31%, 6.2%, respectively, p<0.01). Endotoxin concentrations in dust samples in homes of Conghua were significantly higher (1794 EU per square meter, 10.95 EU endotoxin/g dust) than those of Guangzhou (508.8 EU per square meter, 6.45 EU endotoxin/g dust, p<0.01). Logistic regression analyses showed a positive association between endotoxin levels and PD20-FEV1 (r=0.174, p<0.05), and a negative association (p<0.01) between endotoxin levels and the wheal sizes of skin response to Dermatophagoides pteronyssinus (Der. p) and Dermatophagoides farinae (Der. f).

Plasma endotoxin in horses presented to an equine referral hospital: Correlation to selected clinical parameters and outcomes

Endotoxaemia is frequently presumed on the basis of clinical signs in horses with colic. Measurements of plasma endotoxin (LPS) are rarely made in clinical cases and there is little information on the correlations between this variable, clinical variables and outcomes. To measure LPS levels in plasma of horses presented to the Philip Leverhulme Equine Hospital on admission and daily for up to 4 days and to relate LPS levels to selected clinical parameters, such as heart rate and packed cell volume, and outcomes. Blood samples were collected and stored at -20°C prior to assay of the plasma using a validated kinetic chromogenic Limulus amoebocyte lysate (LAL) assay. Clinical parameters and outcome variables were collected from hospital records. Associations were determined by Chi-squared test and logistic regression analysis. Daily blood samples were collected from 234 horses. LPS was detected in 26.5% of the study population and in 29% of those horses presented for colic. Horses providing samples with detectable LPS were more likely to die whilst in the hospital than those that did not (P = 0.045). Horses presenting with colic were more likely to have detectable LPS in their plasma than noncolic cases (P = 0.037), although LPS was detected in the plasma of 8 out of 42 noncolic horses. A horse that did not meet the study definition of clinical endotoxaemia was 10 times less likely to provide a positive LPS sample (OR 0.10, 95% CI: 0.05-0.22). The proportion of horses providing samples with detectable LPS was similar to other studies. LPS was detected in the minority of horses presented with colic. Increased levels of LPS positively correlated with packed cell volume and with risk of mortality in colic cases.

Influence of sample preservation on endotoxin measurement in water

Appropriate preservation of a range of water types prior to analysis for endotoxin was investigated, including sample storage and addition of sodium thiosulphate. Biologically active endotoxin in water samples was assayed using a chromogenic Limulus Amoebocyte Lysate (LAL) assay. Statistical analysis of measured mean endotoxin levels obtained for samples with and without sodium thiosulphate showed no significant difference in results. There was a 44% mean decline in the concentration of detectable endotoxin in water samples stored at -80 degrees C for 4 weeks compared with samples stored at 4 degrees C and analysed within 24 hours. Freezing of water samples at -80 degrees C in pyrogen-free containers for 4 weeks or longer, then thawing may lead to considerable endotoxin loss; however the addition of sodium thiosulphate to water samples interferes minimally with the LAL assay. These results provide methodological information that can be used to assist researchers in future water endotoxin monitoring studies. The validation and standardisation of water sample preservation protocols are necessary, given the likely increase in the quantification of endotoxin levels in a variety of water sources and the use of such results for health effect determinations.

Influence of selected antimicrobials on the viability, endotoxicity and lipopolysaccharide composition of Pseudomonas aeruginosa in vitro

This research focused on the influence of selected antimicrobial agents (AMAs) on the lipopolysaccharide (LPS) composition of Pseudomonas aeruginosa, a common causative agent of nosocomial infections. As LPS has been shown to play a role in attachment and virulence, the research is primarily aimed at shedding light on the response of these organisms to cleaning regimens in healthcare settings using various disinfectants. The endotoxicity and viability of the organisms following disinfection were further investigated via propagation in sublethal concentrations of the selected AMAs. The AMAs included a CIP chlorinated disinfectant, a heavy-duty alkaline detergent and a phenolic handwash solution. The effects of the antimicrobials on LPS both from intact cells and from debris were assessed by gas chromatography–mass spectrometry (GC–MS) analysis and a chromogenic Limulus amoebocyte lysate assay. Results indicated significant changes in the supramolecular structure of the O-polysaccharide when exposed to the AMAs. Adaptations occurred in both the total assessed saccharide and the lipid fractions, especially in the case of the heavy-duty alkaline detergent. Endotoxicity was found to be influenced by changes in the O-chain rather than the lipid fraction. The phenolic handwash and chlorine-based AMA treatments resulted in a slight decrease in the total amount of fatty acids in the LPS compared with saccharides, whereas the heavy-duty alkaline detergent resulted in a notable reduction in total saccharides. Microbial adaptation of the supramolecular structure of LPS may cause a reduction in membrane solubility of these organisms in an aqueous environment, thus affecting the organism's susceptibility to water-soluble AMAs as well as its ability to adhere to charged surfaces.

Indoor airborne endotoxin assessment in homes of Paris newborn babies

The aims of this study were first to assess airborne endotoxin levels in the dwellings of 162 newborns living in Paris twice during a 1-year period, and second, to identify predictors for endotoxin concentrations using questionnaire data in relation to housing factors and living conditions. Air samples were collected on a glass fiber filter in polystyrene filter holders, using a pump at a flow rate of 3.5 l/min for 24 h placed in the main room of the home. Endotoxin levels were measured using a chromogenic kinetic Limulus Amoebocyte Lysate test. Geometric means (geometric standard deviation) of airborne endotoxin levels at two different visits were respectively 0.509 (4.289) EU/m3 and 0.557 (3.029) EU/m3. Airborne endotoxin levels were significantly increased: (i) in cold season (P = 0.024), with (ii) the presence of visible cockroaches in the previous 12 months at home (P < 0.001), (iii) increased number of inhabitants per square meter (P = 0.012), (iv) the high frequency of cleaning with the floor cloths (P = 0.0014), and (v) the low frequency of vacuuming (P = 0.0045). This study provided for the first time airborne endotoxin levels issued from repeated measurements in Paris dwellings. PRACTICAL IMPLICATIONS This analysis contributed to identify a few factors that determined indoor airborne endotoxin levels. However, the predictive model including housing factors and living conditions poorly estimated endotoxin levels. Consequently, multiple samples and longer sampling periods might improve the estimate of long-term airborne endotoxin exposure especially its variability, in cohort studies.

Effect of root canal procedures on endotoxins and endodontic pathogens

The purpose of this study was to determine the amount of endotoxin (lipopolysaccharide) and cultivable bacteria in human necrotic root canals before (S1) and after chemo-mechanical preparation using chlorhexidine (CHX) gel as auxiliary chemical substance (S2), and after 7 days of intracanal dressing (S3) in order to evaluate the anti-endotoxin and antimicrobial effects of endodontic procedures. Twenty-four teeth were selected for the present study. Chemo-mechanical preparation was performed using 2% CHX gel and three different intracanal medicaments [CaOH2 paste; 2% CHX gel; and CaOH2 + 2% CHX gel]. A quantitative chromogenic Limulus amoebocyte lysate assay was used to measure the amount of endotoxin. Aerobic and anaerobic techniques were used to isolate and identify bacteria, and to determine the bacterial reduction by counting colony-forming units (CFU). Endotoxins and bacteria were present in 100% of the initial samples, with endotoxin concentration ranging from 62.93 to 214.56 UE/ml and CFU ranging from 4 x 10(5) to 2.6 x 10(6). After chemo-mechanical preparation a mean endotoxin reduction of 44.4% was found. Eight (33.3%) root canals were still positive by culture analysis with a mean reduction of bacteria (CFU) of 99.96%. After 7 days of intracanal dressing, endotoxin concentration decreased by only 1.4% compared with S2, and residual bacteria were recovered by culture analysis in 13 cases (54.1%). No significant difference was found among different intracanal medicaments. Relatively high values of endotoxin were still present in the root canal after chemo-mechanical preparation although the majority of bacteria were eliminated. No improvement was achieved by 7 days of intracanal dressing.

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 2 mg/g of cell paste of 95% pure, mono-disperse protein having ⩽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 × 20 μg 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.

Influence of Commercial Sanitizers on Lipopolysaccharide Production by Salmonella Enteritidis ATCC 13076

The effect of typical sanitizers on the composition and toxicity of lipopolysaccharides (LPSs) produced by Salmonella Enteritidis ATCC 13076 was analyzed. Salmonella Enteritidis was propagated up to the late exponential phase in the presence of commercial sanitizing solutions. LPS was extracted and derivatized with trifluoroacetylation, and gas chromatography–mass spectrometry analysis and the chromogenic Limulus amoebocyte lysate assay were used to assess the ultrastructure and toxicity of the LPS. The viability and debris formation during growth were evaluated to verify the bactericidal and bacteriostatic effects of the sanitizers and to assess sanitizer effects on LPS formation. The LPSs produced were quantified at 1.7 × 104, 1.2 × 104, 3.6 × 103, and 9.6 × 104 [KDO]·minus;1 for the controls and the organisms grown in the presence of a chlorinated sanitizer, a heavy-duty alkaline cleaner, and a phenolic hand wash solution, respectively. In response to these treatments, the short-chain polysaccharide fractions of the LPSs in the Salmonella Enteritidis cells increased. This finding suggests that this organism increases the low-molecular-weight fraction of the LPS in relation to the high-molecular-weight fraction to survive these unfavorable conditions. The cumulative change in the LPS in response to the sanitizers influenced the toxicity of the LPS; however, this change could not be related to an individual compound within any of the assessed fractions.

Quantification of endotoxins in necrotic root canals from symptomatic and asymptomatic teeth

The purpose of this investigation was to quantify the concentration of endotoxin in necrotic root canals and investigate the possible relationship between the concentration of endotoxin and endodontic signs and symptoms. Samples were collected from root canals of 50 patients requiring endodontic treatment due to necrosis of the pulpal tissue. Anaerobic techniques were used to determine the number of c.f.u. in each sample. A quantitative chromogenic Limulus amoebocyte lysate assay was used to measure the concentration of endotoxin in each sample. The presence of c.f.u. was detected by culture in all samples (range 10(2)-5x10(6)). In samples from cases of patients with spontaneous pain, the mean c.f.u. was 1.43x10(6) while in asymptomatic cases it was 9.1x10(4). Endotoxin was present in all the samples studied [range 2390.0-22100.0 endotoxin units (EU) ml-1]. The mean concentration of endotoxin in samples from patients with spontaneous pain was 18540.0 EU ml-1 while in asymptomatic cases it was 12030.0 EU ml-1. Asymptomatic cases generally had lower levels of endotoxin (i.e. a negative association). A positive association was found between endotoxin and symptomatic cases (e.g. spontaneous pain, tenderness to percussion, pain on palpation, swelling and purulent exudates). This study showed that endotoxin is present in high concentrations in root canals of symptomatic teeth. There was a positive correlation between the concentration of endotoxin in the root canal and the presence of endodontic signs and symptoms.