Endotoxin Units Research Articles

Functional infrared thermography imaging can be used to assess the effectiveness of Maxicam Gel® in pre-emptively treating transient synovitis and lameness in horses.

Diagnosing and treating lameness in horses is essential to improving their welfare. In equine orthopedic practice, infrared thermography (IRT) can indirectly detect soreness. Non-steroidal anti-inflammatory drugs can treat painful and inflammatory processes in horses. Using IRT, the efficacy of meloxicam (Maxicam Gel®) was evaluated in pre-treating transient synovitis in horses induced by a middle carpal joint injection of lipopolysaccharides (LPS) from E. coli 055:B5 at a dose of 10 endotoxin units. In a cross-over design, six healthy horses were randomly assigned to receive either 0.6 mg/kg of oral Maxicam Gel® (MAXVO) or a mock administration (control group, C) following a two-week washout period. IRT of the middle carpal joint, visual lameness assessment and joint circumference were recorded over time. Clinical and hematological evaluations were performed. Synovial fluid aspirates were analyzed for total nucleated cell count, total protein, and prostaglandin E2. A mixed effects analysis of variance was performed for repeated measures over time, followed by Tukey's test. A multinomial logistic regression was conducted to determine whether there is a relationship between a thermography temperature change and the lameness score. There were no changes in joint circumference. The MAXVO group showed a lower rectal temperature 4 h after synovitis induction. The C group presented an increase in neutrophils and a decrease in total hemoglobin and hematocrit 8 h after induction. No changes were observed in the synovial fluid between groups. The horses that received meloxicam did not show clinically significant lameness at any time, while the C group showed an increase in lameness 2, 4, and 8 h after synovitis induction. IRT indicated that the skin surface temperature of the middle carpal joint was lower in horses who received meloxicam, suggesting a reduction in the inflammatory process induced by LPS. It was observed that the maximum temperature peaks in the dorsopalmar and lateropalmar positions can be utilized to predict the severity of lameness, particularly when the temperature rises above 34°C. Horses pre-treated with meloxicam showed either reduced or no indication of mild to moderate pain and presented a lowehr thermographic temperature, which indicates the effectiveness of Maxicam Gel® as an anti-inflammatory.

Effect of short-term abomasal corn starch infusions on postruminal fermentation and blood measures

It is possible that some of the systemic responses to subacute ruminal acidosis (SARA) may be caused by increased intestinal starch fermentation. The objective of this experiment was to evaluate the effect of abomasal infusion of up to 3 g of corn starch/kg body weight (approximately 1.6 kg of starch/d) on fecal measures of fermentation, plasma acute phase proteins, and white blood cell populations. Six ruminally cannulated cows in late lactation were randomly assigned to duplicate 3 × 3 Latin squares with 21-d periods. Cows were fed a 20.6% starch TMR twice daily and during the last 7 d of each period cows were abomasally infused with corn starch at 0 (CON), 1 (ST1), or 3 (ST3) g/kg body weight split into 2 bolus infusions, provided every 12 h. Fecal samples were collected at 0, 6, 12, and 18 h following feeding on d 21 and were analyzed for pH, VFA, lactic acid, and lipopolysaccharide (LPS). Composite fecal samples were used to estimate apparent total-tract nutrient digestibility using undigested neutral detergent fiber as an internal marker. Blood samples were collected at 0 and 6 h relative to feeding on d 14, 18, and 21 of each period. Concentrations of haptoglobin and serum amyloid A in plasma were measured in all samples, 0 h samples on d 14 and 21 were used to measure white blood cell populations, and 0 h samples from d 14, 18, and 21 were used for flow cytometric analysis of γδ T cells. Data were analyzed in SAS using models that included fixed effects of treatment and period and the random effects of cow and square. For blood measures, d 14 samples collected before the initiation of abomasal infusions were included as covariates. Time (d or h) was added as a repeated measure in variables that included multiple samples during the abomasal infusion period. A contrast was used to determine the linear effect of increasing abomasal corn starch. Abomasal corn starch linearly decreased fecal pH and linearly increased fecal total VFA and LPS, but effects were modest, with fecal pH, total VFA, and LPS changing from 6.96, 57.7 mM, and 4.14 log10 endotoxin units (EU) per gram for the CON treatment to 6.69, 64.1 mM, and 4.58 log10 EU/g for the ST3 treatment, respectively. This suggests that we did not induce hindgut acidosis. There were no effects of treatment on apparent total-tract starch digestibility or fecal starch content (mean of 96.9% and 2.2%, respectively). Treatment did not affect serum acute phase proteins or most circulating white blood cells, but the proportion of circulating γδ T cells tended to linearly decrease from 6.69% for CON to 4.61% for ST3. Contrary to our hypothesis, increased hindgut starch fermentation did not induce an inflammatory response in this study.

Compounding Achromobacter Phages for Therapeutic Applications.

Achromobacter species colonization of Cystic Fibrosis respiratory airways is an increasing concern. Two adult patients with Cystic Fibrosis colonized by Achromobacter xylosoxidans CF418 or Achromobacter ruhlandii CF116 experienced fatal exacerbations. Achromobacter spp. are naturally resistant to several antibiotics. Therefore, phages could be valuable as therapeutics for the control of Achromobacter. In this study, thirteen lytic phages were isolated and characterized at the morphological and genomic levels for potential future use in phage therapy. They are presented here as the Achromobacter Kumeyaay phage collection. Six distinct Achromobacter phage genome clusters were identified based on a comprehensive phylogenetic analysis of the Kumeyaay collection as well as the publicly available Achromobacter phages. The infectivity of all phages in the Kumeyaay collection was tested in 23 Achromobacter clinical isolates; 78% of these isolates were lysed by at least one phage. A cryptic prophage was induced in Achromobacter xylosoxidans CF418 when infected with some of the lytic phages. This prophage genome was characterized and is presented as Achromobacter phage CF418-P1. Prophage induction during lytic phage preparation for therapy interventions require further exploration. Large-scale production of phages and removal of endotoxins using an octanol-based procedure resulted in a phage concentrate of 1 × 109 plaque-forming units per milliliter with an endotoxin concentration of 65 endotoxin units per milliliter, which is below the Food and Drugs Administration recommended maximum threshold for human administration. This study provides a comprehensive framework for the isolation, bioinformatic characterization, and safe production of phages to kill Achromobacter spp. in order to potentially manage Cystic Fibrosis (CF) pulmonary infections.

Plasma lipopolysaccharide elevations in cattle associated with early-stage infection by Fasciola hepatica.

Fasciolosis is an endemic zoonotic parasitic disease with significant impacts on human health and both animal health and production. Early post-infection impacts on the host remain unclear. The objective of this study was to determine the changes, if any, to levels of endotoxin in cattle plasma in response to early-stage infection with Fasciola hepatica. Thirty-six (36) commercial bred cattle were experimentally infected with approximately 400 viable metacercariae. Plasma lipopolysaccharide (endotoxin) levels were examined on 24 occasions from 0 h before infection to 336 h after infection using the Limulus Amoebocyte Lysate chromogenic end point assay and compared with that of six (6) uninfected control animals. Peak lipopolysaccharide levels in infected animals were reached at 52 h after infection and returned to pre-infection levels at time 144 h after infection. Infected animals had significantly elevated lipopolysaccharide levels between 24 and 120 h after infection when compared to uninfected animals. The mean change in endotoxin units (EU)/mL over time after infection was statistically significant in infected animals. Elevations of lipopolysaccharide occurred in all infected animals suggesting a possible repeatable and titratable endotoxemia conducive to therapeutic agent model development.

Occupational Bioaerosol Exposures Associated with Poultry Farming

ABSTRACT This study aimed to investigate occupational exposure to particulate dust, endotoxin, and (1–3)-β-D-glucan among workers involved in various poultry farming activities. A total of 298 personal samples were collected from randomly selected individuals from exposure groups based on distinct poultry farming activities comprising broiler farms, rearing, laying, hatchery, and catching activities. Aside from the inhalable particulate dust concentration that was determined, filter extracts were also analyzed for (1–3)-β-D-glucan and endotoxin using the endpoint Glucatell® and Limulus amoebocyte lysate (LAL) assays, respectively. Data were analyzed using STATA 12 and linear regression models developed. The mean (GM) dust particulate concentration was 11.04 mg/m3 (GSD = 3.87); 2298 endotoxin units (EU/m3) (GSD = 10.56) and 149 ng/m3 for (1–3)-β-D-glucan (GSD = 4.62). A modest positive correlation was observed between log-transformed endotoxin and (1–3)-β-D-glucan concentrations (Pearson r = 0.44, p < .001), whilst a moderate negative correlation was observed for inhalable dust particulate and (1–3)-β-D-glucan (Pearson r = −0.33, p < .001). However, there was a very poor correlation between inhalable dust and endotoxin (Pearson r = −0.02, p < .001). In the regression models, exposure group based on the nature of farming activity explained 50% of the variability in dust particulate and glucan levels. For dust particulate, rearing activities were significant predictors of higher dust levels compared to hatchery work, while rearing, laying, broiler and catching activities were significant predictors of higher endotoxin or glucan levels. Furthermore, working in a small broiler was a significant determinant of elevated glucan exposures. Farms using automated laying activities had significantly higher particulate levels compared to those using manual laying activities. This study revealed that workers engaged in poultry farming activities were exposed to significantly high levels of inhalable particulate dust, endotoxin, and (1–3)-β-D-glucan concentrations, posing an increased risk for adverse respiratory health effects in these farm workers.

Exposure Characterization of Wood Dust Particulate, Endotoxins, and (1-3)-β-d-Glucans, and Their Determinants in Mozambiquan Wood Processing Workers.

Dust generated from wood processing comprises a heterogeneous mixture of inorganic and organic particles, including wood fragments, microorganisms, endotoxins, (1-3)-β-d-glucans, and allergens. This study characterized exposure to wood dust and its determinants in the Mozambiquan wood processing industry. A total of 124 personal inhalable samples, collected from a stratified random sample of 30 workers, were analysed for dust particulate, endotoxins, and (1-3)-β-d-glucans. Mixed-effects models were developed to investigate significant exposure determinants. The geometric mean (GM) inhalable dust particulate concentrations were 3.29 mg m-3, 98 endotoxin units (EU) m-3, and 123 ng m-3 for (1-3)-β-d-glucans. Significant predictors for higher particulate levels included machinery (GMR = 1.93), sawing (GMR = 2.80), carpentry (GMR = 2.77), or painting (GMR = 3.03) tasks. Lebombo-ironwood species was associated with higher dust particulate levels (GMR = 1.97). Determinants of endotoxin concentrations included working with dry wood and damp cleaning methods, which were associated with lower levels. Working in closed buildings (GMR = 3.10) and dry sweeping methods were associated with higher (1-3)-β-d-glucan concentrations (GMR = 1.99). Work tasks in certain exposure groups (machinery, sawing, carpentry, painting), processing certain wood species (Lebombo-ironwood) and working in closed buildings were associated with higher exposures, whilst using dry wood and damp cleaning practices reduced exposure levels.

Histamine promotes adhesion of neutrophils by inhibition of autophagy in dairy cows with subacute ruminal acidosis.

Subacute ruminal acidosis (SARA), a common digestive disease in dairy cows, is accompanied by systemic inflammation and high concentrations of histamine in blood. Histamine-induced neutrophil adhesion may play an important role in the systemic inflammation experienced by cows during SARA. Autophagy, an intracellular degradation system, regulates recycling of membrane-associated integrin and may be involved in histamine-induced adhesion of bovine neutrophils. In the present study, 20 multiparous mid-lactation cows (average body weight 486 ± 24 kg) fitted with ruminal fistula were assigned to a control group (n = 10) or a SARA group (n = 10). We induced SARA by feeding different combinations of wheat-barley pellets and chopped alfalfa hay for 8 wk; SARA was defined as a ruminal pH <5.6 for at least 3 h/d. Blood samples were collected in wk 8. Compared with controls, SARA cows had greater serum concentrations of tumor necrosis factor-α, IL-6, IL-1β, lipopolysaccharide (LPS)-binding protein, haptoglobin, and serum amyloid A. Serum concentrations of these proinflammatory factors had strong positive correlations with the concentration of serum histamine and LPS. In ex vivo adhesion experiments, the number of adherent neutrophils was greater in the SARA group. Additionally, membrane protein abundance of adhesion molecules such as integrin α-L precursor (CD11a) and integrin α-M precursor (CD11b) was greater in neutrophils of the SARA group, confirming enhanced adhesion ability. Neutrophils of SARA cows had greater number of autophagosomes, greater protein abundance of autophagy substrate sequestosome-1 (p62), and higher ratio of microtubule associated proteins 1A/1B light chain 3 (LC3)-II to LC3-I, indicating congestion during the late phase of autophagy flux. For in vitro experiments, neutrophils isolated from control cows were incubated with 0.4 endotoxin units (EU)/mL LPS or 7 μM histamine for 0, 1, 2, and 4 h, respectively. We detected linear and quadratic effects for the number of adherent neutrophils after histamine treatment with a peak response at 2 h, whereas no significant effect was detected after LPS treatment. Membrane protein abundance of CD11a and CD11b was greater after histamine treatment, suggesting that it may have an inhibitory effect on the degradation of adhesion molecules. The greater abundance of p62, higher ratio of LC3-II to LC3-I, and increased co-localization between CD11b and LC3 after histamine treatment indicated that recycling of adhesion molecules and autophagy flux were blocked. These effects were not aggravated further in the presence of chloroquine, a specific inhibitor of the late phase of autophagy flux. Overall, our results revealed that histamine increases adhesion of neutrophils by inhibiting autophagy in dairy cows with SARA.

PD32-10 KIDNEY STONE ENDOTOXIN CONCENTRATION CORRELATES WITH POST-OPERATIVE SEPSIS FOLLOWING PERCUTANEOUS NEPHROLITHOTOMY

You have accessJournal of UrologyCME1 May 2022PD32-10 KIDNEY STONE ENDOTOXIN CONCENTRATION CORRELATES WITH POST-OPERATIVE SEPSIS FOLLOWING PERCUTANEOUS NEPHROLITHOTOMY David T. Tzou, Farhan Anwar, Ava C. Wong, David T. Harris, Thomas Chi, and Gayatri Vedantam David T. TzouDavid T. Tzou More articles by this author , Farhan AnwarFarhan Anwar More articles by this author , Ava C. WongAva C. Wong More articles by this author , David T. HarrisDavid T. Harris More articles by this author , Thomas ChiThomas Chi More articles by this author , and Gayatri VedantamGayatri Vedantam More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002583.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Patients with infected kidney stones are at risk for post-operative sepsis following surgical removal of their calculi. Of all the surgical approaches, percutaneous nephrolithotomy (PCNL) remains associated with the highest risk for experiencing post-operative SIRS (severe inflammatory response syndrome). Accurate prediction of patients at risk for SIRS after surgery would be of clinical value. While previous studies have demonstrated infected kidney stones contain variable levels of endotoxin concentrations, these were limited in their rigor and ability to correlate stone immunobiology with important clinical outcomes. The aim of this study was to quantitate endotoxin levels amongst PCNL patients who experienced and did not experience SIRS. METHODS: Between October 2020 and June 2021, urine & stone specimens from consecutive stone patients were prospectively collected as part of the Registry for Stones of the Kidneys and Ureter (ReSKU) at the University of Arizona. Clinical data including stone analyses and post-operative outcomes have been tracked. Equal weights of stones were uniformly broken using an easy-to-replicate ‘beat-beating’ homogenization approach, allowing for consistent measurement of stone endotoxin using a Pierce™ LAL Chromogenic Endotoxin Quantification Kit (ThermoFisher, Waltham, MA). Endotoxin levels from stones removed during PCNL were compared between patients who experienced and did not experience SIRS. Independent T-test was used to determine statistical significance. RESULTS: Endotoxin levels amongst 4 patients with infection calculi who experienced SIRS were compared to 16 patients who did not experience SIRS – 8 of which had infection calculi and 8 had non-infection calculi (Figure 1). In the SIRS group, the mean Endotoxin concentration was 1.084 endotoxin units (EU)/ml compared to 0.476 EU/ml in the non-SIRS group (p<0.01). CONCLUSIONS: Higher kidney stone bacterial endotoxin concentrations are present in patients who experienced SIRS after percutaneous stone removal compared to those who did not. Whether exceeding a threshold endotoxin level corresponds to patients experiencing SIRS warrants future study. Source of Funding: P30DK081943 – SUBK00015084 - NIH/NIDDK; University of Arizona Health Sciences Career Development Award © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e550 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information David T. Tzou More articles by this author Farhan Anwar More articles by this author Ava C. Wong More articles by this author David T. Harris More articles by this author Thomas Chi More articles by this author Gayatri Vedantam More articles by this author Expand All Advertisement PDF DownloadLoading ...

Comparison of pyrogen assays by testing products exhibiting low endotoxin recovery.

The use of pyrogen tests to assess the risk of endotoxin in biological products has increased recently due to concerns of some regulatory authorities about products exhibiting low endotoxin recovery (LER). Manufacturers increasingly seek to reduce the use of animals unless essential to assure patient safety. The current study compares the ability of the monocyte activation test (MAT) and the bacterial endotoxin test (BET) to the rabbit pyrogen test (RPT) to detect endotoxin spikes in samples of products shown to exhibit LER. Product samples or water were spiked with endotoxin and held for three days or tested immediately in the BET, the RPT, and two variations of the MAT at the same time. Results show high sensitivity to endotoxin of both the BET and MAT, and much lower sensitivity of the RPT, indicating that much higher levels of reference standard endotoxin are required to induce pyrogenicity in the RPT than the 5 endotoxin units (EU) per kg common threshold. The results of the BET and MAT correlated well for the detection of endotoxin spike in water. We also show that LER (masking of endotoxin) found in the BET is also seen in the MAT and RPT, suggesting that the products themselves elicit a biological inactivation of spiked endotoxin over time, thereby rendering it less or non-pyrogenic. We conclude that the non-animal MAT option is a suitable replacement for the RPT to measure spiked endotoxin in biopharmaceuticals.

Metabolic endotoxemia is dictated by the type of lipopolysaccharide.

Lipopolysaccharides (LPSs) can promote metabolic endotoxemia, which is considered inflammatory and metabolically detrimental based on Toll-like receptor (TLR)4 agonists, such as Escherichia coli-derived LPS. LPSs from certain bacteria antagonize TLR4 yet contribute to endotoxemia measured by endotoxin units (EUs). We found that E.coli LPS impairs gut barrier function and worsens glycemic control in mice, but equal doses of LPSs from other bacteria do not. Matching the LPS dose from R.sphaeroides and E.coli by EUs reveals that only E.coli LPS promotes dysglycemia and adipose inflammation, delays intestinal glucose absorption, and augments insulin and glucagon-like peptide (GLP)-1 secretion. Metabolically beneficial endotoxemia promoted by R.sphaeroides LPS counteracts dysglycemia caused by an equal dose of E.coli LPS and improves glucose control in obese mice. The concept of metabolic endotoxemia should be expanded beyond LPS load to include LPS characteristics, such as lipid A acylation, which dictates the effect of metabolic endotoxemia.

A cluster of hypersensitivity pneumonitis associated with exposure to metalworking fluids.

Workers exposed to metalworking fluids (MWF) can develop respiratory illnesses including hypersensitivity pneumonitis (HP). These respiratory manifestations are likely due to microbial contamination of aerosolized MWF. This paper reports a cluster of HP and respiratory symptoms at a manufacturing plant where MWF and workplace air were contaminated with bacterial endotoxin despite frequent negative bacterial cultures of MWF. A pulmonologist assessed and treated three workers with respiratory symptoms. The Occupational Safety and Health Administration (OSHA) inspected the plant. OSHA's investigation included bacterial culture of MWF, measurement of endotoxin concentrations in MWF and workplace air, review of the employer's fluid management program, and distribution of a cross-sectional symptom questionnaire. Three workers had biopsy-confirmed HP. In addition, 30.8% of questionnaire respondents reported work-related respiratory symptoms. OSHA detected endotoxin levels as high as 92,000 endotoxin units (EU)/ml in MWF and 3200 EU/m3 in air. Endotoxin concentrations and risk of MWF inhalation were highest near an unenclosed multistation computer numerical control machine. A contractor had tested this machine's MWF for bacterial growth weekly during the preceding three years, and most (96.0%) of those tests were negative. Contaminated MWF can cause severe occupational lung disease even if microorganisms do not grow in fluid cultures. Endotoxin testing can increase the sensitivity of detection of microbial contamination. However, employers should not rely solely upon MWF testing data to protect workers. Medical surveillance and meticulous source control, such as engineering controls to suppress MWF mist and prevent its inhalation, can reduce the likelihood of respiratory disease.

Miniaturization, Parallelization, and Automation of Endotoxin Detection by Centrifugal Microfluidics.

We demonstrate microfluidic automation and parallelization of Limulus amebocyte lysate (LAL)-based bacterial endotoxin testing using centrifugal microfluidics. LAL is the standard reagent to test for endotoxin contaminations in injectable pharmaceuticals. The main features of the introduced system are more than 90% reduction of LAL consumption, from 100 μL/reaction to 9.6 μL/reaction, automated liquid handling to reduce opportunities for contamination and manual handling errors, and microfluidic parallelization by integrating 104 reactions into a single centrifugal microplate. In a single Eclipse microplate, 21 samples and their positive product controls are tested in duplicate. In addition, a standard curve with up to five points is generated, resulting in a total of 104 reactions. Test samples with a defined concentration of 0.5 endotoxin units per milliliter were tested, resulting in a coefficient of variation below 0.75%. A key feature for achieving a small coefficient of variation is ensuring the same path length along the microfluidic channels to the final reaction chambers for each sample and the reagent, so that any unspecific adsorption to the polymer surfaces does not affect the accuracy and precision. Analysis of a sample containing naturally occurring endotoxin with the developed microfluidic microplate yielded comparable results to the conventional testing method. A test with eight commercially available pharmaceuticals was found to pass all requirements for bacterial endotoxin testing as specified in the United States Pharmacopeia. The automated endotoxin testing system reveals specific advantages of centrifugal microfluidics for analytical biochemistry applications. Small liquid volumes are handled (metered, mixed, and aliquoted) in a very precise, highly integrated, and highly parallel manner within mass-fabricated microplates.

Particulate matter and airborne endotoxin concentration in calf barns and their association with lung consolidation, inflammation, and infection

Agricultural operations are important sources of organic dust containing particulate matter (PM) and endotoxins, which have possible negative health consequences for both humans and animals. Dust concentrations and composition in calf barns, as well as the potential health effects for these animals, are scarcely documented. The objective of this study was to measure PM fractions and endotoxin concentrations in calf barns and study their associations with lung consolidation, respiratory tract inflammation, and infection in group-housed calves. In this cross-sectional study, samples from 24 dairy farms and 23 beef farms were collected in Belgium from January to April 2017. PM1.0, PM2.5 and PM10 (defined as particulate matter passing through a size-selective inlet with a 50% efficiency cut-off at a 1.0-μm, 2.5-μm, and 10-μm aerodynamic diameter, respectively) were sampled during a 24-h period using a Grimm aerosol spectrometer (Grimm Aerosol Technik Ainring GmbH & Co. KG). Endotoxin concentration was measured in the PM10 fraction. Thoracic ultrasonography was performed and broncho-alveolar lavage fluid was collected for cytology and bacteriology. Average PM concentrations were 16.3 µg/m3 (standard deviation, SD: 17.1; range: 0.20-771), 25.0 µg/m3 (SD: 25.3; range: 0.50-144.9), and 70.3 µg/m3 (SD: 54.5; range: 1.6-251.2) for PM1.0, PM2.5, and PM10, respectively. Mean endotoxin in the PM10 fraction was 4.2 endotoxin units (EU)/µg (SD: 5.50; range: 0.03-30.3). Concentrations in air were 205.7 EU/m3 (SD: 197.5; range: 2.32-901.0). Lung consolidations with a depth of ≥1, ≥3, and ≥6 cm were present in 43.1% (146/339), 27.4% (93/339), and 15.3% (52/339) of the calves, respectively. Exposure to fine (PM1.0) PM fractions was associated with increased odds of lung consolidations of ≥1 cm (odds ratio, OR: 3.3; confidence interval (CI): 1.5-7.1), ≥3 cm (OR: 2.8; CI: 1.2-7.1), and ≥6 cm (OR: 12.3; CI: 1.2-125.0). The odds of having lung consolidations of ≥1 cm (OR: 13.9; CI: 3.4-58.8) and ≥3 cm (OR: 6.7; 1.7-27.0) were higher when endotoxin concentrations in the dust mass exceeded 8.5 EU/µg. Broncho-alveolar lavage fluid neutrophil percentage was positively associated with PM10 concentration, and epithelial cell percentage was negatively associated with this fraction. Concentration of PM2.5 was positively associated with epithelial cell percentage and isolation of Pasteurella multocida. Although concentrations of fine dust are lower in calf barns than in poultry and pig housings, in this study they were associated with pneumonia in calves. Dust control strategies for reducing fine dust fractions in calf barns may benefit human and animal respiratory health.

The lipopolysaccharide model for the experimental induction of transient lameness and synovitis in Standardbred horses

An established lipopolysaccharide (LPS) model previously described in Warmbloods, was inconsistent in Standardbred horses, where lameness was not detected despite the presence of synovitis. The present study aimed to determine the dose of LPS from E. coli O55:B5 required to induce mild to moderate lameness following middle carpal joint injection in Standardbred horses and to quantitate the induced lameness over time, with and without anti-inflammatory pre-treatment. In a baseline trial, eight healthy, clinically sound Standardbred horses were used in a rule-based dose-escalation design trial, starting at a dose of 10 endotoxin units (EU). Lameness at trot was evaluated visually and quantitatively (using an inertial-sensor system and pressure plate analysis). Synovial fluid aspirates were analysed for total nucleated cell counts, total protein and prostaglandin E2 (PGE2). Following 2 months wash-out, the effective LPS-dose determined in the baseline trial was used to evaluate the effect of anti-inflammatory treatment. A mixed model for repeated measures with horse as random effect was used for analysis.After injection of 10 EU LPS, the desired degree of lameness was observed in the baseline trial, with maximal lameness at post-injection hour (PIH) 4, followed by a rapid decline and return to baseline by PIH 48. No lameness was observed following pre-treatment with meloxicam. In synovial fluid, PGE2 was significantly higher at PIH 8 and PIH 24 in the baseline trial compared with following meloxicam pre-treatment. In conclusion, injection of the middle carpal joint with 10 EU LPS consistently induces a transient lameness and synovitis in Standardbred horses.

Running at Increasing Intensities in the Heat Induces Transient Gut Perturbations.

The risk of exercise-induced endotoxemia is increased in the heat and is primarily attributable to changes in gut permeability resulting in the translocation of lipopolysaccharides (LPS) into the circulation. The purpose of this study was to quantify the acute changes in gut permeability and LPS translocation during submaximal continuous and high-intensity interval exercise under heat stress. A total of 12 well-trained male runners (age 37 [7]y, maximal oxygen uptake [VO2max] 61.0 [6.8]mL·min-1·kg-1) undertook 2 treadmill runs of 2 × 15-minutes at 60% and 75% VO2max and up to 8 × 1-minutes at 95% VO2max in HOT (34°C, 68% relative humidity) and COOL (18°C, 57% relative humidity) conditions. Venous blood samples were collected at the baseline, following each running intensity, and 1 hour postexercise. Blood samples were analyzed for markers of intestinal permeability (LPS, LPS binding protein, and intestinal fatty acid-binding protein). The increase in LPS binding protein following each exercise intensity in the HOT condition was 4% (5.3μg·mL-1, 2.4-8.4; mean, 95% confidence interval, P < .001), 32% (4.6μg·mL-1, 1.8-7.4; P = .002), and 30% (3.0μg·mL-1, 0.03-5.9; P = .047) greater than in the COOL condition. LPS was 69% higher than baseline following running at 75% VO2max in the HOT condition (0.2 endotoxin units·mL-1, 0.1-0.4; P = .011). Intestinal fatty acid-binding protein increased 43% (2.1ng·mL-1, 0.1-4.2; P = .04) 1 hour postexercise in HOT compared with the COOL condition. Small increases in LPS concentration during exercise in the heat and subsequent increases in intestinal fatty acid-binding protein and LPS binding protein indicate a capacity to tolerate acute, transient intestinal disturbance in well-trained endurance runners.

Endotoxin, Food Allergen Sensitization, and Food Allergy: A Complementary Epidemiologic and Experimental Study

A Tsuang, A Grishin, G Grishina. Allergy. 2020;75(3):625–635 The epidemiologic aspect of the study aimed to describe the association between exposure to house dust endotoxin and sensitization to food allergens, while the experimental component compared the measurement of cytokines secreted from peripheral blood mononuclear cells (PBMCs) after stimulation with endotoxin in allergic and nonallergic children. 6963 participants, ages 1 to 84 years, from the 2005–2006 NHANES were included in the epidemiologic study. Twenty-one children (ages 1–19 years) from pediatric clinics at the Mount Sinai Health System with food allergy to either egg, milk or peanut, and healthy controls were included in the experimental study. Dust endotoxin measurement in the household was …

Effects of lipopolysaccharide exposure on the inflammatory response, butyrate flux, and metabolic function of the ruminal epithelium using an ex vivo model

Acidotic conditions in the rumen have been associated with compromised barrier function of the ruminal epithelium and translocation of microbe-associated molecular patterns (MAMP) such as lipopolysaccharide (LPS). Interaction of MAMP with the ruminal epithelium may also induce a local proinflammatory response. The aim of this study was to evaluate the potential proinflammatory response of the ruminal epithelium following LPS exposure in Ussing chambers, to investigate whether LPS exposure affects the flux and metabolism of butyrate. Ruminal epithelial tissue from 9 Holstein bull calves were mounted into Ussing chambers and exposed to 0, 10,000, 50,000, or 200,000 endotoxin units (EU)/mL LPS for a duration of 5 h. Radiolabeled 14C-butyrate (15 mM) was added to the mucosal buffer to assess the mucosal-to-serosal flux of 14C-butyrate. Additional Ussing chambers, without radioisotope, were exposed to either 0 or 200,000 EU/mL LPS and were used to measure the release of β-hydroxybutyrate (BHB) and IL1B into the buffer, and to collect epithelial tissue for analysis of gene expression. Genes associated with inflammation (TNF, IL1B, CXCL8, PTGS2, TGFB1, TLR2, TLR4), nutrient transport (MCT1, MCT4, SLC5A8, GLUT1), and metabolic function (ACAT1, BDH1, MCU, IGFBP3, IGFBP5) were selected and analyzed using quantitative real-time PCR. Butyrate flux was not significantly affected by LPS exposure; however, we detected a tendency for the mucosal-to-serosal butyrate flux to increase linearly with LPS dose. Bidirectional releases of BHB and IL1B were not affected by LPS exposure. Expression of PTGS2, TGFB1, TLR4, and MCU were downregulated following exposure to LPS ex vivo. We detected no effects on the expression of genes associated with nutrient transport. The results of the present study are interpreted to indicate that, although the inflammatory response of the ruminal epithelium was slightly suppressed, exposure to LPS may have altered metabolic function.

Optimization and bioactivity verification of porcine recombinant visfatin with high expression and low endotoxin content using pig liver as template

In order to obtain the porcine recombinant visfatin protein with high expression and low endotoxin content, the current study aims to express and verify the biological activity of the purified porcine recombinant visfatin protein. Firstly, four different expression strains were successfully constructed. Then they were simultaneously induced at 37 °C for 4 h and 16 °C for 16 h. The results showed that Visfatin-pET28a-Transetta was the best strain with high protein expression and purity at 16 °C induction for 16 h. After that, endotoxin was reduced from the recombinant visfatin until the residual endotoxin was less than one endotoxin units per milliliter (EU/mL). Finally, the purified porcine recombinant visfatin protein was incubated with RAW264.7 cells. The results of cell counting kit-8 (CCK-8) showed the survival rate of the cells first increased and then decreased with the increase in visfatin concentration. When the concentration of visfatin was 700 ng/mL, the survival rate of the cells was the highest. Thereafter, control (PBS), Visfatin and Visfatin + PolymyxinB (Ploy.B) groups were incubated with the RAW264.7 cells for 6 h. Real-time quantitative polymerase chain reaction (RT-qPCR) and Enzyme Linked Immuno-Sorbent Assay (ELISA) results showed that, as compared to the control group, the expressions of interleukin (IL)-1β, tumor necrosis factor (TNF)-α and monocyte chemoattractant protein (MCP)-1 in Visfatin group were significantly increased (P < 0.05). However, there was no significant difference between the Visfatin and Visfatin + Poly.B groups, indicating that porcine recombinant visfatin protein promoted the inflammatory activity of RAW264.7 cells while the residual endotoxin did not play a role, suggesting biological activity of porcine recombinant visfatin protein.

A possible beneficial effect of Bacteroides on faecal lipopolysaccharide activity and cardiovascular diseases

Faecal lipopolysaccharides (LPS) have attracted attention as potent elements to explain a correlation between the gut microbiota and cardiovascular disease (CVD) progression. However, the underlying mechanism of how specific gut bacteria contribute to faecal LPS levels remains unclear. We retrospectively analysed the data of 92 patients and found that the abundance of the genus Bacteroides was significantly and negatively correlated with faecal LPS levels. The controls showed a higher abundance of Bacteroides than that in the patients with CVD. The endotoxin units of the Bacteroides LPS, as determined by the limulus amoebocyte lysate (LAL) tests, were drastically lower than those of the Escherichia coli LPS; similarly, the Bacteroides LPS induced relatively low levels of pro-inflammatory cytokine production and did not induce sepsis in mice. Fermenting patient faecal samples in a single-batch fermentation system with Bacteroides probiotics led to a significant increase in the Bacteroides abundance, suggesting that the human gut microbiota could be manipulated toward decreasing the faecal LPS levels. In the clinical perspective, Bacteroides decrease faecal LPS levels because of their reduced LAL activity; therefore, increasing Bacteroides abundance might serve as a novel therapeutic approach to prevent CVD via reducing faecal LPS levels and suppressing immune responses.

The Type of Bacterial Lipopolysaccharides Dictates Insulin Resistance, GLP-1 and Insulin Secretion During Metabolic Endotoxemia

Lipopolysaccharides (LPS) can promote metabolic endotoxemia, which is usually modelled using Escherichia coli derived LPS, a potent Toll-like receptor (TLR)4 agonist. LPS from other bacteria can antagonize TLR4 and still contribute to Endotoxin Units (EU). We found that LPS from E. coli impaired glycemic control in mice, whereas equal doses of LPS (up to 0.4 mg/kg) from Salmonella minnesota, Porphyromonas gingivalis and Rhodobacter sphaeroides did not alter blood glucose. Matching the LPS dose from R. sphaeroides and E. coli defined in EU (0.9x106 EU/kg) revealed that only E. coli LPS promoted dysglycemia and augmented glucose-stimulated insulin and GLP-1 secretion in mice. R. sphaeroides LPS antagonized dysglycemia caused by an equal dose (0.2 mg/kg) of E. coli LPS. Acute and chronic delivery of R. sphaeroides LPS improved insulin sensitivity in obese mice. We propose that the concept of metabolic endotoxemia should be expanded beyond LPS load (EU) to include LPS characteristics.