Recombinant factor C assay for measuring endotoxin in house dust: comparison with LAL, and (1 --> 3)-beta-D-glucans

BACKGROUND: Measurement of exposure to environmental endotoxin is frequently performed using a Limulus amebocyte lysate (LAL) based assay. Recently, a new method has become available with similar sensitivity and potentially greater specificity using recombinant Factor C (rFC) from the horseshoe crab Carcinoscorpius rotundicauda. A preliminary study was carried out to determine the comparability of LAL and rFC in measuring endotoxins in house dust for large scale epidemiologic studies. METHODS: House dust samples were collected from family rooms by vacuuming 1 m2 of the center of the room. Sixty sieved house dust samples were assayed for endotoxin by LAL (Cambrex, KQCL lysate) and rFC (Pyrogene, Cambrex) and for (1 --> 3)-beta-D-glucans by ELISA. The resistant parallel line estimation was used for data analysis of LAL and rFC. A four-parameter logistic fit with inverse prediction was used to calculate (1 --> 3)-beta-D-glucan levels of the samples. RESULTS: The spike recovery was 113.63% (95% CI = 101.69, 125.57%) for LAL and 99.69% (95% CI = 90.14, 109.24%) for rFC assays. The LAL assay gave higher endotoxin estimates compared with rFC. The LAL and rFC estimates were highly correlated (r = 0.86, P < 0.0001). The difference between LAL and rFC endotoxin estimates correlated with the LAL estimates (r = 0.51, P < 0.0001). However, the difference was not correlated with (1 --> 3)-beta-D-glucans. CONCLUSION: LAL and rFC gave comparable results, hence either assay can be used for studies of endotoxin exposure. The current study shows that (1 --> 3)-beta-D-glucan is not a major factor interfering with endotoxin measurements in house dust using a Cambrex KQCL LAL preparation.     

Effects of alkali-treated proteins: feeding studies with free and protein-bound lysinoalanine in rats and other animals.

To find out whether alkali-treated proteins posses nephrotoxic properties, feeding studies were conducted with drastically treated soybean protein and casein, and also with lysinoalanine (LAL), the amino acid known to be formed in protein subjected to high pH at elevated temperature. The feeding of synthesized LAL to rats at dietary levels of 100 ppm and above induced typical renal changes, called nephrocytomegalia. No such changes or any other indications of toxicity were observed, however, upon feeding much higher levels of LAL (up to 6,000 ppm) when provided as the protein-bound compound in alkali-treated casein or soybean protein. When set free by complete acid hydrolysis, LAL induced considerable renal activity, comparable to that of the synthetic compound. These results indicate that alkali treatment of proteins does not induce nephrotoxic properties provided that the compound remains protein-bound. Some nephrotoxic activity was observed, however, with peptide-boound LAL in break-down products (molecular weight less than 5,000) of alkali-treated casein, but considerably less than that of the free compound. LAL-analyses in blood, urine, and feces of rats fed free or protein-bound LAL indicated a positive correlation between intestinal absorption and nephrotoxic potential. No renal changes were encountered upon feeding diets with 1,000 ppm synthetic LAL to mice, hamsters, rabbits, quail, dogs or monkeys, which suggest a species specificity of LAL-induced renal changes in rats.     

58例输液反应原因分析

目的分析58例临床输液反应的原因,寻找预防措施,减少输液反应的发生。方法对我院近两年发生的58例输液反应从致热原、细菌、药物因素、输液器具、季节、患者年龄等方面进行分析。结果对输液反应的药液进行热原检测,共有5例阳性;血培养7例阳性;药液细菌培养2例阳性,同一批号未开封药液细菌培养无细菌生长;输液器及头皮针细菌培养和热原检测全部阴性;引起输液反应的药物以中药制剂、大分子物质、血液制品及含K 的药物为主;秋季为高发季节;发生率高的为中老年人。结论引起输液反应的原因复杂,严格质量控制和无菌操作,以减少输液反应的发生。     

血液净化装置的体外循环血路的细菌内毒素检测方法探讨

本文通过对鲎试剂的灵敏度复核、干扰试验、并与家兔法试验对照,建立用细菌内毒素法检查血液净化装置的体外循环血路中的热原物质的方法。该方法比家兔法灵敏度高,重现性好。     

Protein-bound D-amino acids, and to a lesser extent lysinoalanine, decrease true ileal protein digestibility in minipigs as determined with (15)N-labeling

Heat and alkali treatment of food may increase the concentrations of protein-bound D-amino acids and cross-links such as lysinoalanine (LAL). To examine how protein treatment affects digestibility, purified test meals [total protein 150 g/kg dry matter (DM), 0.44 MJ/(kg BW(0.75). d)] were prepared, containing (g/kg DM) casein, 75; beta-lactoglobulin, 50; or wheat protein, 40. Each was (15)N-labeled. Test proteins were used either in their native form or after treatment for 6 or 24 h at 65 degrees C, pH 10.5-11.5. Each meal was fed to nine adult miniature pigs (twofold complete cross-classification). Chyme was collected continuously over 33 h postprandially via T-fistulas in the terminal ileum, and digestibilities of test proteins and individual L- and D-amino acids were calculated on the basis of recovery of (15)N and the respective amino acids in the chyme. Treatment of casein, beta-lactoglobulin or wheat protein for 24 h increased levels of D-amino acid residues. L-Asparagine and aspartate (L-Asx) were particularly susceptible; 14. 7 +/- 0.4, 11.7 +/- 0.2 and 11.0 +/- 0.9%, respectively, underwent racemization. LAL levels increased in parallel; 11.7 +/- 0.3, 13.6 +/- 0 and 14.8 +/- 0.0%, respectively, of total lysine was converted to LAL. At the same time, prececal protein digestibility was decreased by 13.4 +/- 2.3, 15.3 +/- 1.4 and 17.8 +/- 1.2% units, respectively (P < 0.05; mean +/- SEM, n = 9). Digestibility of individual L-amino acids decreased by 10-15%, but L-amino acids prone to peptic cleavage, such as L-phenylalanine and L-tyrosine, were not affected. Digestibilities of D-amino acids and LAL were approximately 35%. It seems that mainly D-amino acids, and to a lesser extent LAL, were responsible for lower digestibility by interfering with peptic cleavage.     

Performance of two different Limulus amebocyte lysate assays for the quantitation of fungal glucan

This study examined the response of various forms and sources of glucans toward two different Limulus amebocyte lysate (LAL) methods, the modified LAL, and Glucatell. The glucans studied were curdlan, laminarin, yeast glucan, barley glucan, paramylon, pullulan, pustulan, mannan, and pachyman (as part of the Glucatell kit). Both methods provided largely similar results for each of the glucans; however, the Glucatell method yielded slightly higher responses to certain structures that may not necessarily be of fungal origin, leading to falsely greater positive results. The performance of each method to measure fungal glucan concentration specifically was then assessed.     

Evaluation of endotoxin content of diphtheria-tetanus-acellular pertussis combined (DTaP) vaccines that interfere with the bacterial endotoxin test

Applicability of the endotoxin test to diphtheria-tetanus-acellular pertussis combined (DTaP) vaccines was examined. We found some DTaP vaccines that strongly interfered with Limulus amoebocyte lysate (LAL) activity of endotoxin without affecting lethal activity of endotoxin in D-galactosamine-treated mice. LAL activity that was interfered in such vaccines increased apparently after the treatment with phosphate buffer at 4 degrees C for a week. The DTaP vaccines that interfered with the endotoxin test showed no significant effect on endotoxin activity in inducing tumor necrosis factor-alpha (TNF-alpha) in rabbit peripheral blood. The in vitro TNF-alpha induction assay was, therefore, suggested to be an appropriate assay method for the quantitative detection of the endotoxin activity in DTaP vaccines.     

Solubility of endotoxins from Escherichia coli and Pseudomonas aeruginosa

BACKGROUND: The Limulus amebocyte lysate (LAL) assay may underestimate endotoxins because only soluble endotoxins are determined. The solubility of endotoxins was, therefore, studied in two species of Gram-negative bacteria. METHODS: Cultures were grown in serum broth, cells were harvested by centrifugation and washed in physiological saline. Bacterial suspensions were either filtered through PTFE filters and air dried at room temperature, dried in polypropylene tubes at room temperature in a desiccator, or freeze dried. Samples were extracted with aqueous Tween 20. The soluble and insoluble fractions were dried, methanolysed, and hydroxy-fatty acid methyl esters were determined by gas chromatography (GC) as markers of endotoxins. RESULTS: The solubility of the endotoxins from Escherichia coli and Pseudomonas aeruginosa ranged from 9% to 83%. Species and drying conditions had substantial influence on the solubility. CONCLUSIONS: Endotoxin exposure may be underestimated in environmental samples by the LAL test because a substantial fraction can be non-soluble and is not detected.     

Monocyte-activation test to reliably measure the pyrogenic content of a vaccine: An in vitro pyrogen test to overcome in vivo limitations

Pyrogen content is one of the critical quality attributes impacting the safety of a product, and there is an increasing need for assays that can reliably measure this attribute in vaccines. The Limulus amebocyte lysate (LAL) assay and the rabbit pyrogen test (RPT) are the canonical animal-based pyrogen tests currently used to release vaccines; however, there are several drawbacks associated with these tests when applied to Bexsero, intrinsically pyrogenic product, containing a meningococcal Outer Membrane Vesicle component. While the RPT, as applied to Bexsero at its given dilution, ensures safe vaccine, it is highly variable and prone to false positive results. On the other hand, the LAL assay although quantitative, can detect only endotoxin pyrogens and is not sufficient for monitoring the safety of Bexsero, which contains both LPS and non-endotoxin pyrogens. Being aware of these limitations of the RPT and LAL when applied to Bexsero, the Monocyte Activation Test (MAT) which is sensitive to both endotoxin and non-endotoxin based pyrogens has been developed as an alternative pyrogen test. Here, the development and the validation of a MAT assay adapted from the European pharmacopoeia for Bexsero, is described. The MAT assay is then used for monitoring the safety and consistency of Bexsero vaccines at release, providing great advantages in terms of reduced variability with respect to RPT, reduction of animal use, in line with the 3Rs principle concerning the protection of animals and faster time to market. In addition the correlation of the MAT to the RPT has been demonstrated supporting the replacement of the in vivo method and the potential application of the assay to other intrinsically pyrogenic vaccines.     

培养时间对A群脑膜炎球菌多糖疫苗质量的影响

通过对卫生部北京生物制品研究所1994～1996年A群脑膜炎球菌多糖疫苗生产有关数据的统计分析认为,1996年多糖原液鲎试验（LAL）合格率下降的原因是细菌大罐培养后期菌体自溶,脂多糖（LPS）污染了多糖原液。SDS-PAGE银染法测定多糖原液中LPS含量偏高的结果支持这一结论．     

Lymphocyte-mediated liver injury in alcohol-related hepatitis.

The pathogenesis of alcohol-related liver disease (ALD) remains inadequately explained. Increasing alcohol intake is associated with an increased risk of ALD, but many heavy drinkers develop no liver damage. An explanation for ALD susceptibility requires theories that extend beyond a biochemical understanding of alcohol metabolism. Several hepatic cell populations are involved in the pathogenesis of liver injury. The liver-associated lymphocyte (LAL) response to alcohol intake plus immune stimulation may determine susceptibility to liver damage. We have isolated rat LALs and demonstrated the following: (1) Liver-associated lymphocytes differ from the peripheral blood lymphocyte pool; the CD8:CD4 ratio is higher in the LAL population than in peripheral blood. (2) Tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 production by these cells is suppressed by regular alcohol intake. (3) Tumor necrosis factor-alpha and interleukin-6 production by LALs is increased after parenteral administration of concanavalin A (Con A) and by Con A in in vitro LAL cultures obtained from healthy (control) and ethanol-consuming rats. (4) In vivo stimuli that lead to increased cytokine production by LALs lead, within 12-24 h, to increased hepatocyte necrosis [elevated alanine aminotransferase (ALT) levels] and apoptosis. (5) Liver-associated lymphocytes isolated from ethanol-consuming rats, transferred to non-ethanol-consuming rats, confer on the latter animals an ethanol-consuming response to Con A. (6) Cytokine release by LALs is quantitatively as significant as that from Kupffer cells after exposure to lipopolysaccharide. (7) In co-culture studies inhibition of TNF-alpha activity reduces hepatocyte apoptosis induced in the presence of activated LALs. (8) Finally, nuclear factor-kappa B inhibition decreases production of nitric oxide and TNF-alpha, with an associated reduction in hepatocyte apoptosis. In summary, our study findings support the suggestion that a role for LALs exists in the pathogenesis of alcohol and Con A-mediated liver disease.     

Evaluation of the endotoxin retention capabilities of inline intravenous filters

The capabilities of inline filters to retain bacteria and endotoxin were examined during simulated extended infusions for up to 168 hr. The tested inline filters were the ELD96 (Pall Biomedical Corp) and the IVEX 2 (Millipore Corp). Approximately 1 x 10(8) total cells of Escherichia coli B. were challenged to the upstream site of the filter. The test solution of 5% dextrose in water, 0.9% saline, Paremental A (A basic solution for total parenteral nutrition (TPN), a TPN solution in use in our clinic were infused continuously up to 168 hr and flow rate was maintained at 83 ml/hr. The effluents were analyzed using the Limulus Amebocyte Lysate (LAL) test to detect endotoxin and also passage of the challenged bacteria was tested at 24-hr intervals over 168 hr. The results were as follows: (1) The viability control culture showed the presence of viable bacteria throughout the 168-hr period of the experiment. (2) During the experiments, all filters produced sterile effluents. (3) LAL assay indicated that only the effluents from the ELD96 contained no detectable endotoxin for 168 hr.     

Standardization of whole blood assay for determination of pyrogenic activity in organic dust samples

To characterize bioaerosol exposure at workplaces standardized methods are necessary. Activity of endotoxin, one component of organic dust, can be quantified with the Limulus-Amoebocyte Lysat test (LAL test). Further information with respect to pyrogenic activity of the organic dust can be achieved by measuring cytokine release of human blood after stimulation with the dust or its extract (whole blood assay).The aim of our study was the standardization of the whole blood assay (WBA) while using cryo-preserved human blood (Qualis Laboratorium) and to compare the outcome of the different cytokines determined by incubation of the blood cells with extracts from dust samples collected at various workplaces.Cytokine release (IL-1β, IL-6, IL-8, TNF-α, MCP-1) was measured by ELISA after stimulation of fresh blood from ten donors as well as cryo-preserved human blood. In both cases blood was stimulated with E. coli endotoxin as well as with 30 dust filter extracts from various workplaces. All dust filter extracts were investigated in the WBA using cryo-preserved blood as well as with LAL test.E. coli endotoxin stimulated the release of IL-1β, IL-6, IL-8, TNF-α and MCP-1 in a dose-dependent manner in fresh as well as cryo-preserved human whole blood.200 pg/ml E. coli endotoxin induced maximal cytokine release in cryo-preserved blood (mean value for IL-1β 2509±418 pg/ml; n=13 experiments) whereas fresh blood of single donors reached a maximum release when stimulated with 50 ng/ml endotoxin (mean value of ten donors 1125±553 pg/ml IL-1β).Using cryo-preserved blood the coefficient of variation (CV) regarding the interassay variability was below 21% for all cytokines measured.Regarding 26 dust sample extracts correlation coefficient r² for LAL test and WBA was between 0.90 and 0.93 (Pearson) for IL-1β, IL-6, IL-8 and TNF-α whereas correlation for MCP-1 was lower (r²=0.59).Two dust sample extracts which showed similar reactivity patterns in LAL test as well as in WBA with respect to IL-1β, IL-6, IL-8 and TNF-α could be differentiated by measuring MCP-1.In conclusion, cryo-preserved blood pools are suitable to standardize WBA.Combination of different outcome variables like IL-1β and MCP-1 improve the characterization from the inflammatory potency of workplace related dust samples.     

Airborne endotoxin from indoor and outdoor environments: effect of sample dilution on the kinetic Limulus amebocyte lysate (LAL) assay

Airborne endotoxins in occupational environments are a potential respiratory hazard to individuals. In this study, airborne endotoxins were collected using open-face and button aerosol samplers from inside animal housing units and downwind from agricultural production sites and a wastewater treatment plant. Filter extracts were then diluted to examine the effect of interfering substances on the kinetic Limulus amebocyte lysate (LAL) assay. In most cases, the overall endotoxin concentration was shown to decrease with increasing dilution up to 1000-fold, suggesting the presence of enhancing substances in the filter extracts. This dilution-dependent effect was most prominent in the open-face endotoxin samples, while button samples displayed little effect. Using a joinpoint regression model, it was determined that a dilution factor of 50 to 100 was generally sufficient to eliminate the presence of enhancing substances. After screening the data for dilution dependent effects, the airborne endotoxin concentrations were determined. The highest endotoxin concentrations, ranging from 2841 to 49,066 endotoxin units m(-3) of air, were found inside swine farrowing and finishing barns. Airborne endotoxin concentrations were 10- to 100-fold lower inside a dairy barn and downwind of other agricultural production sites and the wastewater treatment plant. Examination of dilution-dependent effects should be considered essential when utilizing the LAL assay, especially if values are to be used for regulatory purposes.     

The adsorption of endotoxin molecule in a microporous polyethylene hollow fibre membrane.

The microporous polyethylene hollow fibre membrane is capable of adsorbing small-sized lipopolysaccharides (LPS) prepared by sonication dispersion, column chromatography on Sephadex G-75 and filtration through a filter membrane with a nominal pore size of 0.025 micron. Small-sized LPS had a one-thousandth of endotoxin activity as compared to intact LPS, when determined by the Synthetic Chromogenic Substrate method of LAL with a specific endotoxin activity of 73.7 ng/micrograms LPS. Fluorescent microscopy of fluorescein conjugated LPS on a microporous polyethylene hollow fibre showed that fluorescein-LPS was adsorbed through the entire depth of the membrane texture. Accordingly the adsorption capacity of the filter for small-sized LPS was determined as 1.65 mg LPS/3.68 m2 surface/116 mg fibre/module.     

The adsorption of endotoxin molecule in a microporous polyethylene hollow fibre membrane

The microporous polyethylene hollow fibre membrane is capable of adsorbing small-sized lipopolysaccharides (LPS) prepared by sonication dispersion, column chromatography on Sephadex G-75 and filtration through a filter membrane with a nominal pore size of 0.025 micron. Small-sized LPS had a one-thousandth of endotoxin activity as compared to intact LPS, when determined by the Synthetic Chromogenic Substrate method of LAL with a specific endotoxin activity of 73.7 ng/micrograms LPS. Fluorescent microscopy of fluorescein conjugated LPS on a microporous polyethylene hollow fibre showed that fluorescein-LPS was adsorbed through the entire depth of the membrane texture. Accordingly the adsorption capacity of the filter for small-sized LPS was determined as 1.65 mg LPS/3.68 m2 surface/116 mg fibre/module.     

New method for obtaining conjugated vaccines

We describe a new method to obtain conjugates against Neisseria meningitidis serogroups A, B, C, Vibrio cholera, and Salmonella typhi and their immunogenicity in Balb/c mice. The saccharides were activated by basic hydrolysis with the generation of amine groups in the saccharidic chain, and these groups were linked to carboxyl groups of tetanus toxoid by via carbodiimida-mediated reaction. The resultant conjugates were administered to mice for the immunogenicity studies. The pirogenicity of LPS was measured by LAL assay. The anti-saccharide IgG, IgG1, and IgG2a antibodies were evaluated. A significant decrease in the pirogenicity of LPS after basic hydrolysis treatment was observed. The conjugates elicited higher titers of anti-polysaccharides or anti-LPS IgG, IgG1, and IgG2a in conjugates than in unconjugated saccharides. The results indicate that we have a new method for obtaining conjugated vaccines and we have demonstrated that after conjugation there was a change in the responses for all saccharides, from thymus-independent to thymus-dependent responses.     

Endotoxin measurement in house dust using the end-point Limulus amoebocyte lysate method

Endotoxin is a lipopolysaccharide, a part of gram-negative bacteria cell membrane commonly present in general and many occupational environments. This paper describes sample preparation and endotoxin measurement in 16 samples of house dust from urban homes (Zagreb, Croatia) using end-point chromogenic Limulus amoebocyte lysate (LAL) bioassay. House dust was collected on cellulose filters by vacuuming bedroom and living room floors, and was kept frozen until assayed. Samples were extracted from filters with a 0.05% solution of Tween-20 in endotoxin-free water. Serial dilutions of samples were measured in duplicates. The linearity of the standard curve was satisfying (r=0.983), as well as the recovery (92 and 110%) and repeatability (coefficient of variation from 0 to 8.5%). The endotoxin levels found in the house dust samples ranged from 4.8 to 200 EU/mg, with the arithmetic mean of 49.5 EU/mg (standard error of the mean of 12.1 EU/mg), and were in the range of house dust endotoxin values obtained by other authors.     

Correlative measurement of four biological contaminants on cotton lint, and their implications for occupational health

Four biological contaminants of cotton fibers (gram-negative bacterial cells, endotoxin, fungal cells, and (1-3)-beta-D-glucan) were measured in 13 cotton lint samples from international origins, using traditional microbiological spread plating and adaptation of the Limulus amoebocyte lysate (LAL) assay. Correlations were evaluated using Spearman's rank correlation analyses. Contamination levels ranged from 713 +/- 212 to 216,830 +/- 30,413 CFU/g gram-negative bacteria; 281 +/- 29 to 9,250 +/- 820 CFU/g fungal cells; 8.30 +/- 0.89 to 137.89 +/- 21.55 ng/g endotoxin; and 15.96 +/- 5.18 to 2,964.42 +/- 313.90 LAL-reactive units/g glucan. Positive correlations existed between all contaminants; however, they were significant only between fungal cells and glucan (p < 0.05) and between endotoxin and glucan (p < 0.01). The highly significant positive correlation between endotoxin and glucan has implications for the health risk posed by the cotton-production environment, as simultaneous inhalation of these agents may cause or exacerbate lung inflammation.     

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

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