Chromogenic Limulus Amebocyte Lysate Test Research Articles

Endotoxin exposure and changes in short-term pulmonary function among sewage workers.

The inhaled endotoxin is considered as a causative factor in the process of acute bronchial obstruction, which can be measured by a decrease in forced expiratory volume in 1 s (FEV1). The aim of this study was to assess endotoxin exposure among sewage treatment plant workers (STPW) and its effect on across-shift changes in respiratory airflow. A group of 78 STPW from a large sewage treatment plant was studied. Inhalable dust for endotoxin assessment was collected using personal aerosol samplers. Endotoxin was assayed with the kinetic, chromogenic Limulus amebocyte lysate test. Across-shift spirometric measurements were performed on Mondays, after 2-days absence from work, with the use of portable spirometer. The forced vital capacity (FVC), and FEV1 parameters were analyzed. Multifactor regression modeling was performed to determine parameters significantly associated with endotoxin exposure. The concentration of inhalable dust and endotoxin ranged from 0.01-1.38 mg/m3 and 0.68-214 endotoxin units per cubic meter of air (EU/m3), respectively. Endotoxins were characterized with the skewed distribution (arithmetic mean (AM) = 38.8 EU/m3, geometric mean (GM) = 15.4 EU/m3, geometric standard deviation (GSD) = 4.21). Through the use of multifactor analysis, which excluded the main confounders (inhalable dust and smoking habit) it was found that, despite low levels of endotoxin, it had significant impact on the observed across-shift decline in FEV1 (p = 0.044). For this parameter, the regression slope was additionally calculated (r = -0.017, p = 0.071). Relatively low levels of endotoxin among sewage treatment plant workers may cause small, but significant across-shift declines in FEV1. The observed relationship was independent of organic dust concentrations and smoking habit. The respiratory protection should be provided for STPW.

Bone marrow-derived cells for cardiovascular cell therapy: an optimized GMP method based on low-density gradient improves cell purity and function.

BackgroundCardiovascular cell therapy represents a promising field, with several approaches currently being tested. The advanced therapy medicinal product (ATMP) for the ongoing METHOD clinical study (“Bone marrow derived cell therapy in the stable phase of chronic ischemic heart disease”) consists of fresh mononuclear cells (MNC) isolated from autologous bone marrow (BM) through density gradient centrifugation on standard Ficoll-Paque. Cells are tested for safety (sterility, endotoxin), identity/potency (cell count, CD45/CD34/CD133, viability) and purity (contaminant granulocytes and platelets).The aims of the present work were (1) to optimize the cell manufacturing process in order to reduce contaminants and (2) to implement additional assays in order to improve product characterization and evaluate product stability.MethodsBM-MNC were isolated by density gradient centrifugation on Ficoll-Paque. The following process parameters were optimized throughout the study: gradient medium density; gradient centrifugation speed and duration; washing conditions.Differential cell count was performed by an automated hematology cell analyzer. Immunophenotype and cell viability were determined by flow cytometry. Functional hematopoietic and mesenchymal precursors and cells with angiogenic potential were assessed by colony-forming assays, cell invasion capacity by a fluorimetric assay. Sterility was tested using an automated microbial detection system, endotoxin by a kinetic chromogenic Limulus amebocyte lysate test. T-test was used for statistical analysis.ResultsA new manufacturing method was set up, based on gradient centrifugation on low density Ficoll-Paque, followed by 2 washing steps, of which the second one at low speed. It led to significantly higher removal of contaminant granulocytes and platelets, improving product purity; the frequencies of CD34+ cells, CD133+ cells and functional hematopoietic and mesenchymal precursors were significantly increased.The process was successfully validated according to Good Manufacturing Practices.The resulting ATMP mainly consisted of viable MNC including CD34+ and CD133+ cell subsets (2.98% ± 1.90% and 0.83% ± 1.32%, respectively), CD184/CXCR4+ cells (34% ± 15%), CD34+/CD133+/CD309+ endothelial precursors (44 ± 21 in 106 total cells), cells with invasion capacity, functional hematopoietic and mesenchymal precursors, cells with angiogenic potential; it was stable for 20 hours at 10°C.ConclusionsThe methodological optimization described here resulted in a significant improvement of ATMP quality, a crucial issue to clinical applications in cardiovascular cell therapy.

Evaluation of the endotoxin binding efficiency of clay minerals using the Limulus Amebocyte lysate test: an in vitro study

Endotoxins are part of the cell wall of Gram-negative bacteria. They are potent immune stimulators and can lead to death if present in high concentrations. Feed additives, which bind endotoxins in the gastrointestinal tract of animals, could help to prevent their negative impact. The objective of our study was to determine the potential of a bentonite (Bentonite 1), a sodium bentonite (Bentonite 2), a chemically treated smectite (Organoclay 1) and a modified attapulgite (Organoclay 2) to bind endotoxins in vitro. Polymyxin B served as positive control. The kinetic chromogenic Limulus Amebocyte lysate test was adapted to measure endotoxin activity. Firstly, a single sorption experiment (10 endotoxin units/mL (EU/mL)) was performed. Polymyxin B and organoclays showed 100% binding efficiency. Secondly, the adsorption efficiency of sorbents in aqueous solution with increasing endotoxin concentrations (2,450 – 51,700 EU/mL) was investigated. Organoclay 1 (0.1%) showed a good binding efficiency in aqueous solution (average 81%), whereas Bentonite 1 (0.1%) obtained a lower binding efficiency (21-54%). The following absorbent capacities were calculated in highest endotoxin concentration: 5.59 mg/g (Organoclay 1) > 3.97 mg/g (Polymyxin B) > 2.58mg/g (Organoclay 2) > 1.55 mg/g (Bentonite 1) > 1.23 mg/g (Bentonite 2). Thirdly, a sorption experiment in artificial intestinal fluid was conducted. Especially for organoclays, which are known to be unspecific adsorbents, the endotoxin binding capacity was significantly reduced. In contrast, Bentonite 1 showed comparable results in artificial intestinal fluid and aqueous solution. Based on the results of this in vitro study, the effect of promising clay minerals will be investigated in in vivo trials.

A bioassay for determination of lipopolysaccharide in environmental samples

Although the limulus amebocyte lysate (LAL) assay is widely used to determine the concentration of LPS in biological samples, it is known to be susceptible to interference caused by substances of non-bacterial origin. In particular, polysaccharides such as β-glucans and pectic polysaccharides from fungi or plants, respectively, were shown to give higher LPS readings than were actually present in the sample. Here, we describe an assay for the determination of LPS in biological samples based on the stimulation of TLR4/MD2/CD14 transfected HEK293 cells which dose dependently release IL-8 upon stimulation with increasing concentrations of highly purified Escherichia coli LPS. The resulting standard curve is used to determine the LPS concentration in unknown samples. We show that the outcome of the LPS stimulation is not affected by the presence of β-glucans or other environmental substances found in dust extracts. Although, we present evidence that the LPS concentration measured with the kinetic chromogenic LAL test correlates with data from the TLR4 assay, the LAL test displays higher LPS readings. We conclude that the described TLR4 assay is a reliable alternative to assess the concentration of LPS in environmental samples without being influenced by polysaccharides such as β-glucans and other environmental substances found in dust extracts.

Increased intestinal inflammatory response and gut barrier dysfunction in Nrf2-deficient mice after traumatic brain injury

Aim: To explore the role of nuclear factor erythroid 2-related factor 2 (Nrf2) in traumatic brain injury (TBI)-induced intestinal inflammatory response and gut barrier dysfunction in the mice. Methods: Wild-type Nrf2 (+/+) and Nrf2 (−/−)-deficient mice were subjected to a moderately severe weight-drop impact-acceleration head injury. We measured nuclear factor kappa B (NF-κB) by electrophoretic mobility shift assay (EMSA); tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) by enzyme-linked immunosorbent assay (ELISA); intercellular adhesion molecule-1 (ICAM-1) by immunohistochemistry; intestinal permeability by lactulose/mannitol (L/M) test; plasma endotoxin by chromogenic limulus amebocyte lysate test. Results: Intestinal levels of NF-κB, pro-inflammatory cytokines and ICAM-1 in Nrf2 (−/−)-deficient mice were significantly higher compared with Nrf2 (+/+) mice at 24 h after TBI. Furthermore, higher intestinal permeability and plasma level of endotoxin were observed in the Nrf2 (−/−) mice compared with Nrf2 (+/+) mice. Conclusion: Nrf2 plays an important protective role in limiting intestinal inflammatory response and gut barrier dysfunction after TBI.

Endotoxin levels in cow's milk samples from farming and non-farming families — The PASTURE study

Background Children from farming families have less allergies than their peers. Consumption of farm milk or unpasteurized milk has been shown to explain (part of) the farming effect or protect against allergies independent of farming status. Objectives We investigated whether the protective effect of farm milk consumption can be explained by higher levels of bacterial endotoxin in milk. Methods We measured endotoxin in approximately 400 farm milk and shop milk samples from farming and non-farming families, respectively, with the kinetic chromogenic Limulus Amebocyte Lysate test and compared endotoxin levels between groups defined by farming status and type of milk (farm milk/shop milk). Results Endotoxin levels were significantly higher in milk samples from non-farming families compared to farming families [adjusted geometric means ratio (95% confidence interval) = 2.61 (1.53–4.43)]. No significant difference in endotoxin levels was found between shop milk and farm milk samples [adjusted geometric means ratio (95% confidence interval) = 1.56 (0.94–2.58)]. The difference between farming and non-farming families could be explained completely for farm milk and partially for shop milk by storage conditions and temperature during transportation to the fieldworker's home. Conclusion The farming effect and the effect of farm milk consumption cannot be explained by higher levels of endotoxin in milk from farmers and farm milk, respectively.

Effect of propofol on mucous permeability and inflammatory mediators expression in the intestine following traumatic brain injury in rats

Aim: To explore whether propofol could in vivo, suppress intestinal permeability and proinflammatory cytokines in the intestine after traumatic brain injury (TBI). Methods: The rats were randomly divided into four groups including control group with right parietal bone window alone and no brain injury, TBI group, TBI + propofol (5 mg/kg/h), and propofol alone group. All animals were killed 72 h later. Plasma endotoxin was assayed by the chromogenic limulus amebocyte lysate test. Intestinal permeability was quantified using the lactulose/mannitol (L/M) test. TNF-α and IL-6 in the intestine were measured using commercially available enzyme-linked immunoassay Kits. Nuclear factor kappa B (NF-κB) was analyzed by Electrophoretic mobility shift assay (EMSA). Results: No rats died before sacrifice. Plasma endotoxin, L/M ratio, intestinal level of NF-κB, TNF-α, and IL-6 remained at baseline level in controlled animals. TBI promoted a significant increment in plasma endotoxin, L/M ratio, intestinal level of NF-κB, TNF-α, and IL-6. Propofol inhibited those parameters at the dose of 5 mg/kg/h. Conclusion: Propofol has the protective effect on the intestine following traumatic brain injury in rats.

Is the activity of soluble CD14 enhanced following major trauma?

The molecule CD14 acts as a receptor for the protein-bound endotoxin (lipopolysaccharide [LPS]) complex and mediates the cellular effects of LPS. The soluble formation, sCD14, is supposed to neutralize circulating LPS (i.e., LPS antagonist) or transfer LPS effects to endothelial cells (i.e., LPS agonist). To elucidate the release of sCD14 per se in patients with major trauma in the early posttrauma period. Our a priori hypothesis was that sCD14 release depends on the plasma LPS concentration simultaneously measured. In a prospective study, 65 patients with multiple injuries (Injury Severity Score, 9-75) were enrolled. The patients were rescued by the medical helicopter service and directly admitted to our clinics. The plasma concentrations of sCD14 (enzyme immunoassay) and LPS (chromogenic limulus amebocyte lysate test) were analyzed. The first blood sample was collected immediately at the accident site. The following samples were drawn at intervals from 2 hours to daily for 2 weeks. Sixty-one patients survived the observation time. Immediately after trauma, their mean sCD14 level was not different from that of healthy individuals. Two hours later, a pronounced increase of sCD14 was observed and sustained throughout the observation period. Even nonsurvivors showed an increased sCD14 release, but less pronounced. In all patients, plasma LPS levels were elevated during the first 12 hours. Major trauma caused an increased release of sCD14. This elevation, however, was not correlated to LPS levels or to the severity of trauma (estimated by trauma scores). We found no evidence that sCD14 levels are of prognostic value regarding survival. Furthermore, the release of sCD14 did not occur in an LPS-neutralizing manner, but rendered possible an LPS-independent mechanism.

Interleukin-6 plasma levels and tumor size in cardiac myxoma

In a certain subset of patients with cardiac myxoma noncardiac, constitutional signs and symptoms that resemble autoimmune disease are observed, such as hypergammaglobulinemia, fever, autoantibodies, and elevated erythrocyte sedimentation rate (ESR) and white blood cell count. The conditions that determine whether a patient becomes affected by these constitutional signs and symptoms are unknown. Interleukin-6 (IL-6), a multifunctional immunoregulatory cytokine that is involved in B-cell differentiation, T-cell activation, acute phase response, and hematopoiesis, has b e e n found in these patients, a In view of the pleiotropic nature of IL-6 and of cytokines in general we wanted to know whether there are further revealing markers that can be linked to the immunologic features of cardiac myxoma. We studied six patients with nonfamilial cardiac myxoma (ages 44 to 65 years; 1 man, 5 women). Diagnosis was established echocardiographically and confirmed histologically. None of the patients had evidence of systemic infection. Endotoxin levels were measured to rule out asymptomatic infection. All patients gave their informed consent. We analyzed preoperative plasma samples for IL-1/3, IL-1 receptor antagonist (IL-lra), IL-2, soluble IL-2 receptor (slL-2r), IL-6, soluble IL-6 receptor (sIL-6r), IL-8, tumor necrosis factor-alpha (TNF-c0, TNF receptor I (TNF-rI) and I! (TNF-rII), and granulocyte macrophage colony-stimulating factor with commercially available enzyme-linked immunosorbent assay kits (Quantikine, R & D Systems, Minneapolis, Minn.). Endotoxin levels were determined with a modified chromogenic limulus amebocyte lysate test (Chromogenix, M61ndal, Sweden). The normal range of the mediator levels was provided by the manufacturer. It is noteworthy that TNF-c~ values may give false-negative results because of the short half-life of this mediator. Patient data and results are listed in Table I. Patients 1 through 4 did not have constitutional symptoms. Patient 1,

Endotoxin concentration in neutropenic patients with suspected gram-negative sepsis: correlation with clinical outcome and determination of anti-endotoxin core antibodies during therapy with polyclonal immunoglobulin M-enriched immunoglobulins.

We carried out a study in patients with severe neutropenia from hematologic malignancy and suspected gram-negative sepsis to evaluate the clinical significance of endotoxin concentrations in plasma before and during a therapeutic intervention with a human polyclonal immunoglobulin M (IgM)-enriched immunoglobulin preparation (Pentaglobin; Biotest, Dreieich, Germany). Twenty-one patients with acute leukemia or non-Hodgkin's lymphoma entered the study upon the development of clinical signs of gram-negative sepsis and received the IgM-enriched immunoglobulin preparation every 6 h for 3 days (total dose, 1.3 liter with 7.8 g of IgM, 7.8 g of IgA, and 49.4 g of IgG), in addition to standardized antibiotic treatment. Concentrations of endotoxin and IgM and IgG antibodies against lipid A and Re lipopolysaccharide (LPS) in plasma were determined by a modified chromogenic Limulus amebocyte lysate test and semiquantitative enzyme linked immunosorbent assay, respectively, before each immunoglobulin infusion and during the following 25 days. Seventeen patients were endotoxin positive; in five of these patients, gram-negative infection was confirmed by microbiologic findings. Prior to therapy, endotoxemia correlated significantly with the occurrence of fever, and a quantitative correlation between the endotoxin concentration and body temperature was found during the individual course of infection in 8 of the 17 patients. Overall mortality from endotoxin-positive sepsis was 41% (7 of 17) and 64% (7 of 11) in patients with symptoms of septic shock. Nonsurvivors had significantly higher maximum concentration of endotoxin in plasma compared with those of survivors at the first study day (median of 126 versus 34 pg/ml; P < 0.05) and during the whole septic episode (median of 126 versus 61 pg/ml; P < 0.05). In survivors, immunoglobulin therapy resulted in a significant decrease in endotoxin levels in plasma within the initial 18-h treatment period, from a pretreatment median value of 28 pg/ml to a value of 8 pg/ml (P< 0.05). In the seven patients who died from uncontrollable infection, no effect of therapy on endotoxin levels in plasma was observed. IgM and IgG antibodies against lipid A and Re LPS increased significantly under immunoglobulin treatment, with significant correlations between antibodies against lipid A and Re LPS. These data strongly suggest a prognostic significance of the endotoxin levels in plasma and a potential effect of treatment with a polyclonal IgM-enriched immunoglobulin preparation. Further studies are needed to substantiate these findings and to assess the impact on the clinical course by way of a prospective placebo-controlled clinical trial.

Effects of different extraction protocols on endotoxin analyses of airborne grain dusts.

The detection of gram-negative bacterial endotoxins in occupational dusts, specifically those from agricultural environments, is of increasing importance in research on occupational lung disease. In this study, the quantitative chromogenic Limulus amebocyte lysate test for the detection of endotoxins in airborne dusts from spring wheat and oats was examined. Different extraction fluids were tested, as were the effects of time on extraction and of repeated freeze-thaw cycles on the extracts. The data suggest that the chromogenic method can be used effectively in the analysis of environmental dusts or their frozen extracts for endotoxin quantitation. Water appears to be the preferable extraction medium, and the length of extraction time may affect the results.