Endotoxin Units Research Articles

Exposure to Wood Dust, Microbial Components, and Terpenes in the Norwegian Sawmill Industry.

Sawmill workers are exposed to wood dust (a well-known carcinogen), microorganisms, endotoxins, resin acids (diterpenes), and vapours containing terpenes, which may cause skin irritation, allergy, and respiratory symptoms including asthma. The health effects of most of these exposures are poorly understood as most studies measure only wood dust. The present study assessed these exposures in the Norwegian sawmill industry, which processes predominantly spruce and pine. Personal exposures of wood dust, resin acids, endotoxin, fungal spores and fragments, mono-, and sesquiterpenes were measured in 10 departments in 11 saw and planer mills. The geometric mean (GM) and geometric standard deviation (GSD) thoracic exposures were: 0.09 mg m−3 dust (GSD 2.6), 3.0 endotoxin units (EU) m−3 (GSD 4.9), 0.4 × 105 fungal spores m−3 (GSD 4.2), 2 × 105 fungal fragments m−3 (GSD 3.2), and 1560 ng m−3 of resin acids (GSD 5.5). The GM (GSD) inhalable exposures were: 0.72 mg m−3 dust (2.6), 17 EU m−3 (4.3), 0.4 × 105 fungal spores m−3 (3.8), and 7508 ng m−3 (4.4) of resin acids. The overall correlation between the thoracic and inhalable exposure was strong for resin acid (rp = 0.84), but moderate for all other components (rp = 0.34–0.64). The GM (GSD) exposure to monoterpenes and sesquiterpenes were 1105 µg m−3 (7.8) and 40 µg m−3 (3.9), respectively. Although mean exposures were relatively low, the variance was large, with exposures regularly exceeding the recommended occupational exposure limits. The exposures to spores and endotoxins were relatively high in the dry timber departments, but exposures to microbial components and mono-and sesquiterpenes were generally highest in areas where green (undried) timber was handled. Dust and resin acid exposure were highest in the dry areas of the sawmills. Low to moderate correlation between components (rp ranging from 0.02 to 0.65) suggests that investigations of exposure–response associations for these components (both individually and combined) are feasible in future epidemiological studies.

Analysis of IL-6 and IL-1β release in cryopreserved pooled human whole blood stimulated with endotoxin.

To overcome the lack of availability of fresh human whole blood for pyrogen detection, we explored the feasibility of utilizing cryopreserved pooled human blood to detect the responses of the pro-inflammatory cytokines IL-6 and IL-1β to LPS. Whole blood was obtained from five donors and incubated with LPS. The quantities of pro-inflammatory cytokines were measured using ELISA, and the results were compared among the samples. After the blood was cryopreserved with Dimethyl sulfoxide (DMSO) (10% v/v) and stored for 4 mo at –196℃, the detection limits of the IL-6/IL-1β responses to LPS were 0.2/0.4 endotoxin units (EU)/ml, respectively, and IL-6/IL-1β release increased in response to LPS in a dose-dependent manner. When these experiments were performed in three separate laboratories, the within-laboratory reproducibility of the IL-6/IL-1β responses was 100%/86.7%, 93.3%/100%, and 86.7%/80%, and the inter-laboratory reproducibility was 92.9%/85.7%, 64.3%/63.6%, and 57.1%/66.7%, respectively. The sensitivity (the probability of correctly classifying positive samples) and specificity (the probability of correctly classifying negative samples) of the IL-6/IL-1β tests were 81.7%/82.5% and 100%/100%, respectively. The results of this study suggest that cryopreserved pooled blood is a convenient and viable alternative for evaluating in vitro pyrogenicity. Additionally, maintaining cryopreserved pooled blood promotes safety for the user because it is released only after pretesting for infection parameters and has lower variation than fresh donations from a variety of donors.

Hydroxyapatite chromatographic procedures for phospholipids: Endotoxin purification methods

ABSTRACTWe studied the one-step purification of endotoxin by hydroxyapatite chromatography using total lipids extracted from Escherichia coli by the Bligh and Dyer method. High endotoxic activity was detected in a fraction eluted from 0.5 mg total lipids using an isopropanol gradient under salt-free buffer conditions. The fraction contained at least 7,000 endotoxin units and was completely free from other phospholipids. Notably, the running time of the chromatographic separation was approximately 4 h, whereas purification by the conventional hot phenol–water procedure requires ≥60 h. The procedure developed here is a simple, scalable, and rapid method for the purification of endotoxin.

Subacute ruminal acidosis affects fermentation and endotoxin concentration in the rumen and relative expression of the CD14/TLR4/MD2 genes involved in lipopolysaccharide systemic immune response in dairy cows

The first objective of this study was to investigate the effects of subacute ruminal acidosis (SARA) on fermentation, ruminal free lipopolysaccharides (LPS), and expression of the cluster of differentiation 14 (CD14), toll-like receptor 4 (TLR4), and myeloid differentiation protein 2 (MD2) complex in white blood cells involved in the systemic immune response in dairy cows. The second objective was a study of whether increased expression of the LPS receptor complex led to increases in the concentrations of plasma high-density lipoprotein (HDL) and serum Ca. Three hundred five dairy cows located in 13 Polish high-yielding dairy commercial farms were selected according to their days in milk (40-150 d; average = 75), 305-d milk yield (10,070-12,041 kg; average = 10,940), and number of lactations (primiparous, n = 139 and multiparous, n = 166). Next, the herds were segregated into 3 groups based on the percentages of cows with an assigned value of ruminal fluid pH: SARA-positive, SARA-risk, and SARA-negative herds. Moreover, 305 selected dairy cows were divided according to the classification based on ruminal fluid pH into 3 groups as healthy (pH >5.81), risk (pH 5.8-5.6) and acidotic cows (pH <5.6). Rumen fluid samples were collected via rumenocentesis. In the AC group, we recorded higher concentrations of ruminal free LPS [4.57 Log10 endotoxin units (EU)/mL; 42,206 EU/mL] compared with the healthy group (4.48 Log10 EU/mL; 34,179 EU/mL). Similarly, the concentration of ruminal free LPS was higher in SARA-positive herds (4.60 Log10 EU/mL; 43,000 EU/mL) compared with SARA-negative herds (4.47 Log10 EU/mL; 32,225 EU/mL). The relative mRNA abundance of genes associated with the function of LPS receptors, such as CD14, TLR4, and MD2, in white blood cells differed between all experimental groups on both cow and herd levels. In the acidotic group, we recorded higher concentrations of HDL (78.16 vs. 68.32 mg/dL) and serum amyloid A (10.80 vs. 9.16 µg/mL) and lower concentrations of Ca (8.26 vs. 10.16 mg/dL) and haptoglobin (470.19 vs. 516.85 ng/mL) compared with the healthy group. Similar results were obtained in the SARA herd status analysis, but the concentration of lipopolysaccharide-binding protein differed statistically. Moreover, the pH of ruminal fluid was negatively correlated with relative mRNA abundance of genes such as CD14, TLR4, MD2, and concentrations of serum HDL and serum amyloid A, although positively correlated with serum Ca. The results indicated that decreases in ruminal fluid pH increased the release of free LPS into the rumen and stimulated the expression of the LPS receptor complex and immune response. Moreover, an increase in the expression of the LPS receptor led to higher concentrations of plasma HDL and lower serum Ca, which may be a protective mechanism against endotoxemia. However, the biological significance of these results needs to be investigated further in larger field trials.

Periodontal-induced chronic inflammation triggers macrophage secretion of Ccl12 to inhibit fibroblast-mediated cardiac wound healing.

Chronic inflammatory diseases, such as periodontal disease, associate with adverse wound healing in response to myocardial infarction (MI). The goal of this study was to elucidate the molecular basis for impaired cardiac wound healing in the setting of periodontal-induced chronic inflammation. Causal network analysis of 168 inflammatory and extracellular matrix genes revealed that chronic inflammation induced by a subseptic dose of Porphyromonas gingivalis lipopolysaccharide (LPS) exacerbated infarct expression of the proinflammatory cytokine Ccl12. Ccl12 prevented initiation of the reparative response by prolonging inflammation and inhibiting fibroblast conversion to myofibroblasts, resulting in diminished scar formation. Macrophage secretion of Ccl12 directly impaired fibronectin and collagen deposition and indirectly stimulated collagen degradation through upregulation of matrix metalloproteinase-2. In post-MI patients, circulating LPS levels strongly associated with the Ccl12 homologue monocyte chemotactic protein 1 (MCP-1). Patients with LPS levels ≥ 1 endotoxin units (EU)/ml (subseptic endotoxemia) at the time of hospitalization had increased end diastolic and systolic dimensions compared with post-MI patients with < 1 EU/ml, indicating that low yet pathological concentrations of circulating LPS adversely impact post-MI left ventricle (LV) remodeling by increasing MCP-1. Our study provides the first evidence to our knowledge that chronic inflammation inhibits reparative fibroblast activation and generates an unfavorable cardiac-healing environment through Ccl12-dependent mechanisms.

Determination and risk assessment of airborne endotoxin concentrations in a university campus

Endotoxin is a cell-wall component of Gram-negative bacteria and is a hazardous biological substance that exerts adverse health effects. However, research on the determination and risk assessment of airborne endotoxins in university campuses is limited. In this study, the levels of airborne endotoxins were measured at different sites of a university campus in China. The arithmetic means for airborne endotoxin levels were 72 endotoxin units (EU)/m3 (range = 37.9–97.6EU/m3). The airborne endotoxin levels varied at different sites and were typically high in laboratories (wastewater control and resource recovery laboratories) and school hospitals. Education environments had higher dose rates and risks (e.g., office) than recreational living places (e.g., gyms). The dose rates and health risks of airborne endotoxins followed the order children > adult male > adult female. Compared with skin contact, inhalation was the main way of airborne endotoxin exposure. No statistical differences were observed among the different sampling times on the same day. Both concentrations of total volatile organic compound and PM2.5 show positive correlations with airborne endotoxins, whereas culturable bioaerosol concentration, temperature, and relative humidity, were less significantly correlated with airborne endotoxin levels (p < 0.05).

A cell‐based approach to quantifying endotoxin in human plasma samples

Poor dietary choices are thought to contribute to the risk of developing a variety of chronic diseases, in part, through promoting a state of low‐grade inflammation. For example, there are multiple research reports indicating that high‐fat meals promote a transient post‐prandial inflammatory state. Several investigators have hypothesized that high fat meals promote the absorption of microbial endotoxin across the gut wall and that once in the circulation this promotes inflammation through the activation of TLR4 signaling pathway on circulating innate immune cells (e.g., monocytes). Direct assessment of endotoxin in human blood samples is problematic. Reliance on the limulus amebocyte lysate (LAL) test for quantifying endotoxin in a protein‐rich matrix, such as human plasma, requires that samples be heated prior to the analyses. Recovery of endotoxin spiked into plasma samples prior to heating is remarkably low, typically less than 1%. This phenomena, commonly referred to as neutralization, raises grave doubts about the suitability of the LAL test for quantifying endotoxin in the human circulation. Many researchers have long recognized the need for a more robust and accurate method for quantifying low levels of endotoxin in human circulation. In 1995, a cell‐based bioassay approach referred to as monocyte activation test (MAT) was developed and marketed as an alternative to LAL test. Unfortunately, the MAT requires that blood samples are processed within a few hours of collection, which greatly limits the utility of this alternative approach. We have developed and tested a cell‐based bioassay (CBA) for quantifying endotoxin in human plasma following storage at −80°C by using THP‐1 cells (i.e., a human monocytic cell line) stably‐transfected with a luciferase reporter driven by multiple NF‐kB promoters. Our CBA provides reliable and reproducible quantitation of endotoxin in unheated human plasma samples with a limit of detection approaching 0.05 endotoxin units (EU)/mL of plasma. Sensitivity and precision of our CBA compares well with the performance of a recently released commercial cell line (InvivoGen). In conclusion, both of these CBAs appear to provide the research community with a reliable alternative to the LAL test for quantifying endotoxin in human plasma.Support or Funding InformationThis research was supported in part by Grant Number P50AT006273 from the National Center for Complementary and Integrative Health (NCCIH), the Office of Dietary Supplements(ODS), and the National Cancer Institute (NCI). Additional funding was provided by the Food‐for‐the‐21st Century Program ‐Nutrition Cluster, which is part of the College of Agriculture, Food and Natural Resources at the University of Missouri ‐ Columbia. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS, NCCIH, ODS, NCI, or the National Institutes of Health.

Bioaerosols in the Suburbs of Ostrava during a One Year Period.

The aim of this paper is to provide information about the concentrations of airborne bioaerosols (airborne bacteria, fungi and endotoxins) in outdoor suburban environments in Ostrava, Moravian-Silesian region, Czech Republic. The methods were based on systematic bioaerosol monitoring during one calendar year, subsequent analysis of the samples and statistical processing. The regression, correlation analysis and analysis of variance for one factor and pairwise comparisons were performed on bioaerosol data to determine their dependence on season, daytime, temperature, humidity and dew point. The results show higher fungi concentrations especially in summer (corrected mean 365 colony forming units - CFU per m3) compared to other seasons (75-209 CFU/m3) and higher concentrations of bacteria in the evening (380 CFU/m3) compared to other parts of the day and seasons (in summer 206-252 CFU/m3 and in winter 81-87 CFU/m3). Concentrations of endotoxins were relatively low throughout the year, on average 0,056 endotoxin units (EU) per m3. The concentration of bioaerosol (bacteria, fungi and endotoxins) were found in ambient air at substantially lower levels than in an indoor environment. Although the concentrations of this bioaerosol greatly fluctuate with temperature, dew point, season and daytime, they do not represent increased health risks.

An Exploratory Investigation of Endotoxin Levels in Novice Long Distance Triathletes, and the Effects of a Multi-Strain Probiotic/Prebiotic, Antioxidant Intervention.

Gastrointestinal (GI) ischemia during exercise is associated with luminal permeability and increased systemic lipopolysaccharides (LPS). This study aimed to assess the impact of a multistrain pro/prebiotic/antioxidant intervention on endotoxin unit levels and GI permeability in recreational athletes. Thirty healthy participants (25 males, 5 females) were randomly assigned either a multistrain pro/prebiotic/antioxidant (LAB4ANTI; 30 billion CFU·day−1 containing 10 billion CFU·day−1 Lactobacillus acidophilus CUL-60 (NCIMB 30157), 10 billion CFU·day−1 Lactobacillus acidophillus CUL-21 (NCIMB 30156), 9.5 billion CFU·day−1 Bifidobacterium bifidum CUL-20 (NCIMB 30172) and 0.5 billion CFU·day−1 Bifidobacterium animalis subspecies lactis CUL-34 (NCIMB 30153)/55.8 mg·day−1 fructooligosaccharides/ 400 mg·day−1 α-lipoic acid, 600 mg·day−1 N-acetyl-carnitine); matched pro/prebiotic (LAB4) or placebo (PL) for 12 weeks preceding a long-distance triathlon. Plasma endotoxin units (via Limulus amebocyte lysate chromogenic quantification) and GI permeability (via 5 h urinary lactulose (L): mannitol (M) recovery) were assessed at baseline, pre-race and six days post-race. Endotoxin unit levels were not significantly different between groups at baseline (LAB4ANTI: 8.20 ± 1.60 pg·mL−1; LAB4: 8.92 ± 1.20 pg·mL−1; PL: 9.72 ± 2.42 pg·mL−1). The use of a 12-week LAB4ANTI intervention significantly reduced endotoxin units both pre-race (4.37 ± 0.51 pg·mL−1) and six days post-race (5.18 ± 0.57 pg·mL−1; p = 0.03, ηp2 = 0.35), but only six days post-race with LAB4 (5.01 ± 0.28 pg·mL−1; p = 0.01, ηp2 = 0.43). In contrast, endotoxin units remained unchanged with PL. L:M significantly increased from 0.01 ± 0.01 at baseline to 0.06 ± 0.01 with PL only (p = 0.004, ηp2 = 0.51). Mean race times (h:min:s) were not statistically different between groups despite faster times with both pro/prebiotoic groups (LAB4ANTI: 13:17:07 ± 0:34:48; LAB4: 12:47:13 ± 0:25:06; PL: 14:12:51 ± 0:29:54; p > 0.05). Combined multistrain pro/prebiotic use may reduce endotoxin unit levels, with LAB4ANTI potentially conferring an additive effect via combined GI modulation and antioxidant protection.

A comparison of the immunological potency of Burkholderia lipopolysaccharides in endotoxemic BALB/c mice.

Lipopolysaccharide is one of the virulence factors of the soil-borne pathogens Burkholderia pseudomallei, B. thailandensis, B. cenocepacia and B. multivorans, which cause septic melioidosis (often in B. pseudomallei infections but rarely in B. thailandensis infections) or cepacia syndromes (commonly in B. cenocepacia infections but rarely in B. multivorans infections). The inflammatory responses in Burkholderia LPS-induced endotoxemia were evaluated in this study. Prior to induction, the conserved structures and functions of each purified LPS were determined using electrophoretic phenotypes, the ratios of 3-hydroxytetradecanoic to 3-hydroxyhexadecanoic acid and endotoxin units. In an in vitro assay, cytokine expression of myeloid differentiation primary response gene 88 and Toll/IL-1 receptor domain containing adapter-inducing INF-β-dependent signaling-dependent signaling differed when stimulated by different LPS. Endotoxemia was induced in mice by s.c. injection as evidenced by increasing serum concentrations of 3-hydroxytetradecanoic acid and the septic prognostic markers CD62E and ICAM-1. During endotoxemia, splenic CD11b+ I-A+ , CD11b+ CD80+ , CD11b+ CD86+ and CD11b+ CD11c+ subpopulations increased. After induction with B. pseudomallei LPS, there were significant increases in splenic CD49b NK cells and CD14 macrophages. The inflamed CD11b+ CCR2+ , CD11b+ CD31+ , CD11b+ CD14+ , resident CD11b+ CX3 CR1+ and progenitor CD11b+ CD34+ cells showed delayed increases in bone marrow. B. multivorans LPS was the most potent inducer of serum cytokines and chemokines, whereas B. cenocepacia LPS induced relatively low concentrations of the chemokines MIP-1α and MIP-1β. Endotoxin activities did not correlate with the virulence of Burkholderia strains. Thus factors other than LPS and/or other mechanisms of low activity LPS must mediate the pathogenicity of highly virulent Burkholderia strains.

Impact of biomass burning on soil microorganisms and plant metabolites: A view from molecular distributions of atmospheric hydroxy fatty acids over Mount Tai

AbstractBiomass burning events (BBEs) in the North China Plain is one of the principal sources of airborne pollutants in China and also for the neighboring countries. To examine the impact of BBEs on soil bacteria and other higher plant metabolites, their tracer compounds, hydroxy fatty acids (FAs), were measured in the bulk particulate matter (total suspended particles (TSP)) over Mount Tai during the period of wheat residue burning in June 2006. Higher inputs of epicuticular waxes and soil microorganisms during high BBEs (H; 6–14 and 27 June) relative to low BBEs (L; 15–26 and 28 June) were characterized by increased concentrations of homologous series of α‐(C9–C32), β‐(C9–C32), and ω‐(C12–C28) hydroxy FAs in TSP samples. However, their relative abundances were not significantly different between H‐BBEs and L‐BBEs, suggesting their common source/transport pathways. We also found higher concentrations of trehalose and mannitol (tracers of soil microbes), and levoglucosan (tracer of biomass combustion) during H‐BBEs than L‐BBEs. These results are consistent with hydroxy FAs, suggesting that they are associated with biomass combustion processes of agricultural wastes as well as re‐suspension of mineral dust and plant pathogens. In addition, enhanced concentrations of endotoxin and mass loading of Gram‐negative bacteria during H‐BBEs (117 endotoxin units (EU) m−3 and 390 ng m−3, respectively) were noteworthy as compared to those in L‐BBEs (22.5 EU m−3 and 75 ng m−3, respectively). Back trajectory analysis and fire spots together with temporal variations of hydroxy FAs revealed an impact of biomass burning on emissions and atmospheric transport of bacteria and plant metabolites.

Endotoxin hitchhiking on polymer nanoparticles

The control of microbial infections is critical for the preparation of biological media including water to prevent lethal septic shock. Sepsis is one of the leading causes of death in the United States. More than half a million patients suffer from sepsis every year. Both gram-positive and gram-negative bacteria are responsible for septic infection by the most common organisms i.e., Escherichia coli and Pseuodomonas aeruginosa. The bacterial cell membrane releases negatively charged endotoxins upon death and enzymatic destruction, which stimulate antigenic response in humans to gram-negative infections. Several methods including distillation, ethylene oxide treatment, filtration and irradiation have been employed to remove endotoxins from contaminated samples, however, the reduction efficiency remains low, and presents a challenge. Polymer nanoparticles can be used to overcome the current inability to effectively sequester endotoxins from water. This process is termed endotoxin hitchhiking. The binding of endotoxin on polymer nanoparticles via electrostatic and hydrophobic interactions offers efficient removal from water. However, the effect of polymer nanoparticles and its surface areas has not been investigated for removal of endotoxins. Poly(ε-caprolactone) (PCL) polymer was tested for its ability to effectively bind and remove endotoxins from water. By employing a simple one-step phase separation technique, we were able to synthesize PCL nanoparticles of 398.3 ± 95.13 nm size and a polydispersity index of 0.2. PCL nanoparticles showed ∼78.8% endotoxin removal efficiency, the equivalent of 3.9 × 105 endotoxin units (EU) per ml. This is 8.34-fold more effective than that reported for commercially available membranes. Transmission electron microscopic images confirmed binding of multiple endotoxins to the nanoparticle surface. The concept of using nanoparticles may be applicable not only to eliminate gram-negative bacteria, but also for any gram-positive bacteria, fungi and parasites.

Occupational Exposure to Bioaerosols in Norwegian Crab Processing Plants.

Aerosolization of components when processing king crab (Paralithodes camtschaticus) and edible crab (Cancer pagurus) may cause occupational health problems when inhaled by workers. A cross-sectional study was carried out in three king crab plants and one edible crab plant. Personal exposure measurements were performed throughout work shifts. Air was collected for measurement of tropomyosin, total protein, endotoxin, trypsin, and N-acetyl-β-d-glucosaminidase (NAGase). T-tests and ANOVAs were used to compare the levels of exposure in the different plants and areas in the plants. Total protein and tropomyosin levels were highest in the edible crab plant, endotoxin levels were highest in king crab plants. King crab exposure levels were highest during raw processing. Tropomyosin levels were highest during raw king crab processing with geometric mean (GM) 9.6 versus 2.5ng m(-3) during cooked processing. Conversely, edible crab tropomyosin levels were highest during cooked processing with GM 45.4 versus 8.7ng m(-3) during raw processing. Endotoxin levels were higher in king crab plants than in the edible crab plant with GM = 6285.5 endotoxin units (EU) m(-3) versus 72 EU m(-3). In the edible crab plant, NAGase levels were highest during raw processing with GM = 853 pmol4-methylumbelliferone (MU) m(-3) versus 422 pmol4-MU m(-3) during cooked processing. Trypsin activity was found in both king crab and edible crab plants and levels were higher in raw than cooked processing. Differences in exposure levels between plants and worker groups (raw and cooked processing) were identified. Norwegian crab processing workers are exposed to airborne proteins, tropomyosin, endotoxins, trypsin, and NAGase in their breathing zone. Levels vary between worker groups and factories.

Exposure to high endotoxin concentration increases wheezing prevalence among laboratory animal workers: a cross-sectional study.

BackgroundEndotoxin from Gram-negative bacteria are found in different concentrations in dust and on the ground of laboratories dealing with small animals and animal houses.MethodsCross-sectional study performed in workplaces of two universities. Dust samples were collected from laboratories and animal facilities housing rats, mice, guinea pigs, rabbits or hamsters and analyzed by the “Limulus amebocyte lysate” (LAL) method. We also sampled workplaces without animals. The concentrations of endotoxin detected in the workplaces were tested for association with wheezing in the last 12 months, asthma defined by self-reported diagnosis and asthma confirmed by bronchial hyperresponsiveness (BHR) to mannitol.ResultsDust samples were obtained at 145 workplaces, 92 with exposure to animals and 53 with no exposure. Exposed group comprised 412 subjects and non-exposed group comprised 339 subjects. Animal-exposed workplaces had higher concentrations of endotoxin, median of 34.2 endotoxin units (EU) per mg of dust (interquartile range, 12.6–65.4), as compared to the non-exposed group, median of 10.2 EU/mg of dust (interquartile range, 2.6–22.2) (p < 0.001). The high concentration of endotoxin (above whole sample median, 20.4 EU/mg) was associated with increased wheezing prevalence (p < 0.001), i.e., 61 % of workers exposed to high endotoxin concentration reported wheezing in the last 12 months compared to 29 % of workers exposed to low endotoxin concentration. The concentration of endotoxin was not associated with asthma report or with BHR confirmed asthma.ConclusionExposure to endotoxin is associated with a higher prevalence of wheezing, but not with asthma as defined by the mannitol bronchial challenge test or by self-reported asthma. Preventive measures are necessary for these workers.

Comparison of lipopolysaccharides and soluble CD14 measurement between clinically endotoxaemic and nonendotoxaemic horses.

Clinically useful biomarkers are needed for early identification of endotoxaemic horses. Soluble CD14 (sCD14) is amplified early in response to inflammatory signals, including bacterial lipopolysaccharide (LPS), and may prove a useful biomarker for clinical endotoxaemia. The aim of this study was to determine if sCD14 could serve as a more reliable biomarker of the clinical signs of endotoxaemia, compared to measuring LPS alone. Prospective observational study in horses at a veterinary teaching hospital. Plasma samples were collected from 20 healthy horses and 35 horses presenting for emergency evaluation. Horses were classified as clinically endotoxaemic, using previously established criteria, if they had a heart rate >70 beats/min, packed cell volume >45% and/or a lesion likely to result in endotoxaemia. Soluble CD14 was measured using a cytometric bead-based assay and LPS was measured using a Limulus amoebocyte lysate (LAL) assay. Soluble CD14 was higher in horses classified as clinically endotoxaemic (median 1102 ng/ml, interquartile range 439 ng/ml), compared to clinically nonendotoxaemic (median 692 ng/ml, interquartile range 455 ng/ml, P = 0.03. There was no difference in LPS concentrations between clinically nonendotoxaemic (median 5.4 endotoxin units [EU]/ml, interquartile range 5 EU/ml) and endotoxaemic horses (median 7.2 EU/ml, interquartile range 17 EU/ml, P = 0.2). There was no correlation between sCD14 and LPS values in paired serum samples. LPS and sCD14 values were used to generate a receiver operating characteristic curve. The area under the curve for LPS and sCD14 was <0.7, suggesting that sCD14 and LPS were poor predictors of clinical endotoxaemia for the horses in this study. Further investigation is warranted to assess the utility of sCD14 measurement as a clinically useful biomarker to identify endotoxaemia in horses.

Gut Microbiota and Metabolic Endotoxemia in Young Obese Mexican Subjects

Background: The gut microbiota plays an important role in human metabolism; previous studies suggest that the imbalance can cause a metabolic endotoxemia that may be linked to weight gain and insulin resistance. The purpose of this study was to investigate the relationship between the gut microbiota composition, the lipopolysaccharide levels and the metabolic profile in obese and normal-weight young subjects. Methods: We studied 32 obese (BMI ≥ 30 kg/m²) and 32 normal-weight subjects (BMI = 18.5-24.9 kg/m²), aged 18-25 years. Quantification of intestinal bacteria was performed by real-time PCR. Endotoxin units were determined with the test QCL-1000, and biochemical profile was performed under a standard protocol of Spinreact. Results: Obese individuals had a BMI of 34.5 (32.9-36.45) kg/m², increased triglycerides (123 vs. 70 mg/dl), total cholesterol (168 vs. 142 mg/dl), and LDL-cholesterol (114 vs. 96.5 mg/dl). In obese subjects body temperature was higher than in normal-weight subjects. We found a greater number of Clostridum leptum and Lactobacillus (p < 0.001) and lower numbers of Prevotella and Escherichia coli (p < 0.001) in the obese group. A decrease of E. coli was associated with an increased risk of lipopolysaccharide levels ranging from 1 to 1.3 EU/ml. A positive correlation was found between serum lipopolysaccharides and BMI (r = 0.46, p = 0.008), triglyceride levels (r = 0.44, p = 0.011) as well as waist circumference (r = 0.34, p = 0.040), being more evident in young obese females. Conclusion: Subclinical metabolic endotoxemia determined by serum concentration of lipopolysaccharides was related to the smallest amount of E. coli, high triglyceride levels, and central adiposity in obese young persons.

Impact of Saccharomyces cerevisiae fermentation product and subacute ruminal acidosis on production, inflammation, and fermentation in the rumen and hindgut of dairy cows

The effects of a Saccharomyces cerevisiae fermentation product (SCFP) on microbial fermentation in the digestive tract, milk production, and inflammation response were determined in Holstein dairy cows under normal and subacute ruminal acidosis (SARA) conditions. Eight lactating dairy cows with ruminal and cecal cannulas were used in a cross-over design with 5 wk experimental periods. During the first experimental period, cows were randomly assigned to treatment, i.e. SCFP (Original XPC, Diamond V, Cedar Rapids, IA) or control. During the second experimental period, treatments were reversed. Experimental periods were separated by a two-week wash out period. During each period, the 4 cows on the SCFP treatment were supplemented with 14g/d of SCFP in 126g of ground corn, whereas the other four cows received 140g ground corn only. During the first 4 wk of each experimental period, all cows received a basal diet. During week 5 of both experimental periods, a SARA challenge was conducted in all cows by replacing 208g/kg of the basal diet with pellets of ground wheat and barley (50:50 on a weight basis). This SARA challenge increased the duration of rumen pH below 5.6 from 11.1 to 311.1min/d, indicating successful induction of SARA. Inducing SARA reduced milk fat from 3.24 to 2.82%, and lowered the acetate to propionate ratio from 3.07 to 1.74 in rumen fluid, from 5.13 to 4.57 in cecal digesta, and from 4.81 to 4.34 in feces. Induction of SARA increased milk protein content from 3.23 to 3.32%, without a concomitant effect on milk protein yield. Inducing SARA also increased endotoxic lipopolysaccharide (LPS) in rumen fluid, cecal digesta and feces from 15,389 to 123,296 endotoxin units (EU)/mL, from 31,982 to 77,380EU/mL, and from 29,679 to 77, 371EU/mL, respectively. Concentrations of LPS, serum amyloid A, haptoglobin, and LPS-binding protein increased by 215.0, 250.6, 181.6 and 47.5%, respectively, in peripheral blood when SARA was induced. The SCFP did not affect feed intake, yields of milk, milk fat, and milk protein but it reduced the variation in rumen pH during control feeding. During the SARA challenge, SCFP tended to reduce ruminal LPS at 6h after feed delivery, increased milk fat percentage (2.71 vs 2.92%, control vs SCFP), and reduced the extent of SARA-associated inflammation response as indicated by a reduction in the concentration of the pro-inflammatory cytokine tumor necrosis factor α (0.09 vs 0.03ng/mL, control vs SCFP). This shows that SCFP can attenuate some of the impact of SARA on production and health of dairy cows.

Assessing endotoxins in equine-derived snake antivenoms: Comparison of the USP pyrogen test and the Limulus Amoebocyte Lysate assay (LAL)

Snake antivenoms are parenterally administered; therefore, endotoxin content must be strictly controlled. Following international indications to calculate endotoxin limits, it was determined that antivenom doses between 20 mL and 120 mL should not exceed 17.5 Endotoxin Units per milliliter (EU/mL) and 2.9 EU/mL, respectively. The rabbit pyrogen test (RPT) has been used to evaluate endotoxin contamination in antivenoms, but some laboratories have recently implemented the LAL assay. We compared the capability of both tests to evaluate endotoxin contamination in antivenoms, and we found that both methods can detect all endotoxin concentrations in the range of the antivenom specifications. The acceptance criteria of RPT and LAL must be harmonized by calculating the endotoxin limit as the quotient of the threshold pyrogenic dose and the therapeutic dose and the dose administered to rabbits as the quotient of the threshold pyrogenic dose and the endotoxin limit. Since endotoxins from Gram-negative bacteria exert different pyrogenicity, if contamination occurred, antivenom batches that induce pyrogenic reactions may be found in spite of passing LAL specifications. Although LAL assay can be used to assess endotoxin content throughout the antivenom manufacturing process, we recommend that the release of final products be based on the results of both methods.

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.

Characterization and Purification of Glutathione S-Transferase from the Liver and Gill Tissues of Ağrı Balık Lake Trout Salmo trutta labrax and the Effects of Heavy Metal Ions on Its Activity.

Glutathione S-transferase (GST) from the liver and gill tissues of Ağrı Balık Lake Trout (also known as Black Sea Trout) Salmo trutta labrax was characterized and purified, and the toxic effects of some heavy metal ions on the enzyme's activity were analyzed. Liver GST was purified 930 times, resulting in 56% yield using glutathione-agarose affinity chromatography and a specific activity of 60.87 endotoxin units (EU)/mg protein. Using the same procedure, gill GST was purified 576 times, resulting in a 60% yield and specific activity of 46.8 EU/mg protein. The purity check of the purified enzymes was determined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Optimal pH, ionic strength, and stable pH were found for each tissue, and separate KM and Vmax values were determined for reduced glutathione and 2,4-dinitrochlorobenzene substrates. Heavy metal ions that have toxic effects on living organisms and are known to contribute to environmental pollution were selected, and their in vitro effects on enzyme activity were analyzed. The IC50 values and Ki constants of those metal ions showing an inhibitory effect on GST activity were determined.