Limulus Amoebocyte Lysate Assay Research Articles

Role of insulin during the development of oligofructose (OF)-induced equine laminitis

Abstract Horses (n = 20) were divided into 2 groups: oligofructose (OF)-induced equine laminitis group (group OF; n = 11) which received 10 g/kg b.w. of OF dissolved in 4 L water via nasogastric intubation, and control group (NS; n = 9) which received 4 L of saline. Blood was collected at 4 h intervals over 72 h study period and analysed by ELISA, kinetic limulus amoebocyte lysate assay, and glucose-oxidase methods. The level of insulin changed significantly in horses which received OF (P < 0.01); there was a significant negative correlation between the level of adiponectin and insulin over time. The results suggested that insulin may play an important role in the development of OF-induced equine laminitis by altering the level of endothelin-1 and nitric oxide.

Endotoxemia Analysis by the Limulus Amoebocyte Lysate Assay in Different Mammal Species Used in Metabolic Studies

Introduction: Lipopolysaccharides (LPS) or so-called endotoxins are potent pro-inflammatory compounds. LPS can be present in the bloodstream in case of septic conditions, leading to measure endotoxemia that is the activity of LPS in plasma. Recent research also reveals a low-grade or so-called metabolic endotoxemia associated with metabolic diseases (e.g. obesity, type 2 diabetes, cardiovascular diseases). In this context, research studies use different experimental models, mostly in humans and rodents. Pig is now emerging as a new animal model in nutritional and metabolic studies. However, information is lacking to date about optimal dilution to be used for sample preparation for the Limulus Amebocyte Lysate test, according to species, especially for pig. Methods: strong>Endotoxemia was measured using the LAL standard reference method. We describe the method of sample preparation and the LAL technique to measure endotoxemia in 4 mammal species: human, mouse, rat and pig. Results: Plasma dilution is necessary to overcome interferences leading to erroneously low or high results. Optimal dilution to avoid interferences in most samples, while maintaining a satisfactory sensitivity, was found to be at least 1/10 for human vs 1/40 for mice, while much higher dilution was mandatory for pigs, namely 1/200. Altogether, mean plasma endotoxemia in all tested samples using the optimal dilution for each species was 0.73 ± 0.05 EU/mL in humans (n=903), 0.9 ± 0.2 EU/mL in rodents (n=295) and 8.5 ±1.3 EU/mL in pigs (n=186), regardless of fasting or postprandial state and/or type of dietary intervention. Conclusion: We show that the LAL assay can be used to determine endotoxemia in fasting and postprandial blood samples from different mammal species including pig. Because its detection is made difficult by interference from other plasma constituents, an essential parameter to overcome this difficulty is the dilution factor that depends on the studied species.

Endotoxin detection in end-stage kidney disease

AimsEndotoxin detection assays are not validated for use in end-stage kidney disease (ESKD). We investigated the accuracy and precision of the kinetic turbidimetric Limulus amoebocyte lysate (LAL) assay to detect...

Determinants of house dust, endotoxin, and β-(1→3)-D-glucan in homes of Danish children.

Little is known about the geographic variation and determinants of bacterial endotoxin and β-(1,3)-D-glucan in Danish house dust. In a population of 317 children, we: (i) described loads and concentrations of floor dust, endotoxin, and β-(1→3)-D-glucan and (ii) their correlations and (iii) assessed their determinants; (iv) Finally, we compared our findings with previous European studies. Bedroom floor dust was analyzed for endotoxin content by the kinetic limulus amoebocyte lysate assay and for β-(1→3)-D-glucan by the inhibition enzyme immunoassay. The parents answered questions regarding potential determinants. We found: geometric means (geometric standard deviations) 186 mg/m(2) (4.3) for dust; 5.46 × 10(3) EU/m(2) (8.0) and 31.1 × 10(3) EU/g (2.6) for endotoxin; and 142 μg/m(2) (14.3) and 0.71 × 10(3) μg/g (7.3) for β-(1→3)-D-glucan. High correlations (r > 0.75) were found between floor dust and endotoxin and β-(1→3)-D-glucan loads, while endotoxin and β-(1→3)-D-glucan concentrations were moderately correlated (r = 0.36-0.41) with the dust load. Having a carpet was positively associated with dust load and with endotoxin and β-(1→3)-D-glucan concentrations. Pet keeping, dwelling type, and dwelling location were determinants of endotoxin concentrations. No other determinants were associated with β-(1→3)-D-glucan concentrations. Compared with other European studies, we found lower β-(1→3)-D-glucan loads and concentrations but higher endotoxin loads and concentrations suggesting a geographically determined different composition of Danish floor dust compared with other European regions.

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

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

Endotoxemia in end-stage kidney disease.

Chronic unexplained inflammation remains a prevalent and clinically significant problem for patients with end-stage kidney disease (ESKD), especially in the dialysis population. The causes of persistent inflammation are likely to be multifactorial, but the underlying mechanisms remain to be elucidated. Endotoxins are reported to play a significant role in the pathogenesis of inflammation in patients with ESKD. However, blood endotoxin measurement with the Limulus amoebocyte lysate (LAL) assay is difficult with current detection systems. The reported degree and prevalence of endotoxemia varies in the literature. There are questions as to whether endotoxemia is truly present; whether the varied findings are due to methodological issues with the LAL assay and whether any endotoxemia that might be present plays a role in chronic inflammation frequently observed in ESKD patients. This review will discuss the challenges of accurate blood endotoxin detection, the potential source of blood endotoxins, and the significance of endotoxemia to patient with ESKD.

Bacterial toxin-inducible gene expression of cathelicidin-B1 in the chicken bursal lymphoma-derived cell line DT40: Functional characterization of cathelicidin-B1

Chicken cathelicidin-B1 (chCATH-B1) is a major host defense peptide of the chicken bursa of Fabricius (BF). To investigate the mechanisms of chCATH-B1 gene expression in the BF, we focused on the DT40 cell line derived from chicken bursal lymphoma as a model for analysis. A cDNA encoding chCATH-B1 precursor was cloned from DT40 cells. The nucleotide sequence of the cDNA was identical with that of the BF chCATH-B1. A broth dilution analysis showed that the synthetic chCATH-B1 exhibited a significant defensive activity against both Escherichia coli and Staphylococcus aureus. A scanning microscopic analysis demonstrated that chCATH-B1 inhibited bacterial growth through membrane destruction with formation of blebs and spheroplasts. Limulus amoebocyte lysate assay and electromobility shift assay results revealed that chCATH-B1 bound to lipopolysaccharide (LPS) and lipoteichoic acid (LTA), which are the surface substances of the E. coli and S. aureus cell, respectively. A chemotactic assay results revealed that chCATH-B1 showed mouse-derived P-815 mastocytoma migrating activity dose-dependently but with a higher concentration, resulting in a loss of the activity. A semi-quantitative real-time RT-PCR analysis revealed that LPS stimulated chCATH-B1 gene expression in a dose-dependent manner and that the LPS-inducible chCATH-B1 gene expression was inhibited by the administration of dexamethasone. The chCATH-B1 mRNA levels in DT40 cells were also increased by the administration of bacterial LTA. The results indicate that bacterial toxins induce chCATH-B1 gene expression in the chicken BF and the peptide expressed in the organ would act against pathogenic microorganisms not only directly but also indirectly by attracting mast cells.

Exposure to Beta-(1,3)-D-glucan in house dust at age 7-10 is associated with airway hyperresponsiveness and atopic asthma by age 11-14.

BackgroundMould exposure has been linked to childhood asthma and bronchial hyper-responsiveness. Few studies have assessed beta-(1,3)-d-glucan (beta-glucan), a significant fungal cell wall constituent, in relation to asthma in adolescence.ObjectiveTo determine whether house dust-derived beta-glucan exposure at age 7–10 is associated with the development and persistence of atopic and non-atopic asthma, and bronchial hyper-responsiveness (BHR) by age 11–14.MethodsDust samples were collected from the 1995 Study of Asthma, Genes, and Environment (SAGE) birth cohort. This cohort was derived from Manitoba provincial healthcare administrative records of children high and low risk for asthma. Samples were collected from the homes of 422 children at age 7–10 and analyzed using beta-glucan and endotoxin-specific Limulus Amoebocyte Lysate assays. Asthma, atopy, and BHR status of each child were also assessed at ages 7–10 and 11–14.ResultsAt age 7–10, beta-glucan dust levels in the home were associated with persistent atopic asthma at age 11–14 (OR 1.79 for each unit increase in levels, 95% CI 1.14–2.81), independent of endotoxin exposure, and Alternaria or Cladosporium sensitization. The likelihood of BHR almost doubled with unit increases in dust beta-glucan in asthmatic children. In children without asthma, exposure to high beta-glucan levels at age 7–10 also elevated risk for BHR in adolescence (OR 1.74, 95% CI 1.05–2.89). New-onset atopic asthma was twice more likely following high beta-glucan exposure in children without asthma but the association did not reach statistical significance. No associations were evident with concurrent asthma phenotype at age 7–10 or non-atopic asthma at age 11–14.ConclusionThese findings implicate home beta-glucan exposure at school-age as a risk factor for persistent atopic asthma and new-onset BHR. The higher prevalence of BHR in urban adolescents may be propagated by this home exposure.

Exposure to Airborne Endotoxins among Sewer Workers: An Exploratory Study

Exploratory bioaerosol sampling was performed in order to assess exposure to airborne endotoxins during sewer work. Personal samples were collected in underground sewer pipes using 37-mm closed-face cassettes containing fibreglass filters (CFC-FG method) or polycarbonate filters (CFC-PC method). Endotoxins were quantified using the limulus amoebocyte lysate assay. Concentrations of airborne endotoxins at sewer workplaces (16-420 EU m(-3)) were higher than those measured outside the sewer network (0.6-122 EU m(-3)). Sewer worker exposure to airborne endotoxins depended on the workplace and on the tasks. Exposure levels were the highest for tasks involving agitation of water and matter, especially for 'chamber cleanup' and 'pipes cleanup' with a high-pressure water jet. Airborne endotoxin levels at the workplace tended to be higher when CFC-FG was used as the sampling method rather than CFC-PC. The adjusted mean of the measured concentrations for CFC-PC represents 57% of the mean observed with CFC-FG. The number of samples collected in the descriptive study was too low for drawing definitive conclusions and further exposure investigations are needed. Therefore, our exploratory study provides new exposure data for the insufficiently documented sewer working environment and it would be useful for designing larger exposures studies.

Clinical investigation of the effect of calcium hydroxide intracanal dressing on bacterial lipopolysaccharide reduction from infected root canals

The purpose of this clinical study was to determine the effect of 7 day intracanal dressing with calcium hydroxide on the amount of bacterial lipopolysaccharide (LPS; endotoxin) in human teeth with necrotic and infected pulp and apical periodontitis. Twenty-five single-rooted teeth with necrotic pulps and apical periodontitis were selected. Samples were collected before (S1), after root canal preparation (S2) and after 7 day intracanal dressing with calcium hydroxide (S3). The limulus amoebocyte lysate assay was used to quantify LPS. LPS was present in 100% of the root canals before (S1), after preparation (S2) and after 7 day intracanal dressing (S3). A significant reduction, equal to 29.54%, was found after root canal preparation (P < 0.05). A significant difference (equal to 25.26% reduction) was also detected between S2 and S3 (P < 0.05). Total endotoxin reduction (S3 compared with S1) was found to be 47.34%. Endotoxin concentration of the infected root canals was reduced after root canal preparation and also after 7 days of dressing of canals with calcium hydroxide; however, relatively high values of endotoxin remained in the root canals.

Circulating CD4+ and CD8+ T cells are activated in inflammatory bowel disease and are associated with plasma markers of inflammation

Inflammatory bowel disease (IBD) is characterized by damage to the gut mucosa and systemic inflammation. We sought to evaluate the role of chronic inflammation on circulating T-cell activation in human subjects with Crohn's disease and ulcerative colitis. We studied 54 patients with IBD and 28 healthy controls. T-cell activation and cycling were assessed in whole blood samples by flow cytometry. Levels of lipopolysaccharide (LPS) were measured in serum by Limulus amoebocyte lysate assay, and plasma levels of inflammatory markers and LPS-binding proteins were measured by ELISA. The proportions of circulating CD4(+) and CD8(+) T lymphocytes in cycle (Ki67(+) ) are increased in patients with IBD compared with these proportions in controls. CD8(+) T cells from patients with IBD are also enriched for cells that expressed CD38 and HLA-DR, and proportions of these cells are related to plasma levels of interleukin-6 and C-reactive protein in these patients. Intracellular interleukin-2 and interferon-γ levels were elevated in resting and polyclonally activated CD4(+) and CD8(+) T cells in patients with IBD when compared with levels from healthy controls. Surprisingly, we did not find increased levels of LPS in the serum of patients with IBD. We did, however, find a signature of recent microbial translocation, as levels of LPS-binding protein are increased in the plasma of patients with IBD compared with plasma levels in healthy controls; LPS-binding protein levels are also directly related to proportions of CD38 HLA-DR-expressing CD4(+) and CD8(+) T cells. Local damage to the gastrointestinal tract in IBD may result in systemic inflammation and T-cell activation.

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

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

Distinct Renal Pathology and a Chemotactic Phenotype after Enterohemorrhagic Escherichia coli Shiga Toxins in Non-Human Primate Models of Hemolytic Uremic Syndrome

Enterohemorrhagic Escherichia coli cause approximately 1.5 million infections globally with 176,000 cases occurring in the United States annually from ingesting contaminated food, most frequently E. coli O157:H7 in ground beef or fresh produce. In severe cases, the painful prodromal hemorrhagic colitis is complicated by potentially lethal hemolytic uremic syndrome (HUS), particularly in children. Bacterial Shiga-like toxins (Stx1, Stx2) are primarily responsible for HUS and the kidney and neurologic damage that ensue. Small animal models are hampered by the inability to reproduce HUS with thrombotic microangiopathy, hemolytic anemia, and acute kidney injury. Earlier, we showed that nonhuman primates (Papio) recapitulated clinical HUS after Stx challenge and that novel therapeutic intervention rescued the animals. Here, we present detailed light and electron microscopic pathology examination of the kidneys from these Stx studies. Stx1 challenge resulted in more severe glomerular endothelial injury, whereas the glomerular injury after Stx2 also included prominent mesangiolysis and an eosinophilic inflammatory infiltration. Both toxins induced glomerular platelet-rich thrombi, interstitial hemorrhage, and tubular injury. Analysis of kidney and other organs for inflammation biomarkers showed a striking chemotactic profile, with extremely high mRNA levels for IL-8, monocyte chemoattractant protein 1, and macrophage inflammatory protein 1α and elevated urine chemokines at 48hours after challenge. These observations give unique insight into the pathologic consequences of each toxin in a near human setting and present potential pathways for therapeutic intervention.

Comparison of inflammatory responses in human cells caused by lipopolysaccharides from Escherichia coli and from indigenous bacteria in aquatic environment

The endotoxic activities of lipopolysaccharides (LPSs) in water samples are usually determined using a Limulus amoebocyte lysate (LAL) assay, but it is known that the determined activities do not always represent their inflammatory potency in humans. In this investigation, the inflammatory responses in three different human cells stimulated with Escherichia coli LPS, keratinocyte, CD14+ monocyte, and THP-1, were compared using cytokine secretion as biomarkers to develop novel in vitro assay systems for detecting changes in inflammatory potencies of endotoxins in aquatic environment. Only THP-1 with 6-h stimulation showed dose-dependent responses in the range of normal endotoxin levels in aquatic environment. Then, the inflammatory potency of environmental LPS, which was purified from river water, was tested using THP-1. The levels and patterns of cytokine secretion after the environmental LPS stimulation were completely different from E. coli LPS. Interleukin 8 (IL-8) secretions after the environmental LPS stimulation were approximately 10-fold higher than those after E. coli LPS stimulation. The environmental LPS also induced much higher levels of TNF-α secretions in THP-1. These results suggest that a diversity of LPS structures in aquatic environment could contribute to stronger and different inflammatory responses. This investigation indicated that the proposed THP-1 assay system could be useful for detecting the changes in inflammatory potencies caused by aquatic bacteria.

Pseudoalteromonas strains are potent immunomodulators owing to low-stimulatory LPS

Many species of marine bacteria elicit a weak immune response. In this study, the aim was to assess the immunomodulatory properties of Gram-negative Pseudoalteromonas strains compared with other marine Gram-negative bacteria and to identify the molecular cause of the immunomodulation. Using murine bone-marrow derived dendritic cells (DCs), it was found that Pseudoalteromonas strains induced low cytokine production and modest up-regulation of surface markers CD40 and CD86 compared with other marine bacteria and Escherichia coli LPS. Two strains, Ps. luteoviolacea and Ps. ruthenica, were further investigated with respect to their immunomodulatory properties in DCs. Both inhibited IL-12 and increased IL-10 production induced by E. coli LPS. LPS isolated from the two Pseudoalteromonas strains had characteristic lipid A bands in SDS-PAGE. Stimulation of HEK293 TLR4/MD2 cells with the isolated LPS confirmed the involvement of LPS and TLR4 and established Pseudoalteromonas LPS as TLR4 antagonists. The isolated LPS was active in the endotoxin limulus amoebocyte lysate assay and capable of inducing increased endocytosis in DCs. This study highlights that antagonistic LPS from Pseudoalteromonas strains has potential as a new candidate of therapeutic agent capable of modulating immune responses.

The influence of probiotic supplementation on gut permeability in patients with metabolic syndrome: an open label, randomized pilot study

Obesity and metabolic disorders are linked to inflammation via gut microbiota and/or gut permeability. Gut-derived endotoxin triggers inflammation leading to metabolic syndrome (MetS) and contributing to oxidative stress. We intended to investigate the effect of Lactobacillus casei Shirota on gut permeability, presence of endotoxin and neutrophil function in MetS. Patients with MetS were randomized to receive 3 × 6.5 × 10⁹ CFU L. casei Shirota (probiotic group) or not for 3 months. Gut permeability was assessed by a differential sugar absorption method and by determination of diaminooxidase serum levels, endotoxin by an adapted limulus amoebocyte lysate assay, neutrophil function and toll-like receptor (TLR) expression by flow cytometry and ELISA was used to detect lipopolysaccharide-binding protein (LBP) and soluble CD14 (sCD14) levels. Twenty-eight patients and 10 healthy controls were included. Gut permeability was significantly increased in MetS compared with controls but did not differ between patient groups. None of the patients were positive for endotoxin. LBP and sCD14 levels were not significantly different from healthy controls. High-sensitive C-reactive protein and LBP levels slightly but significantly increased after 3 months within the probiotics group. Neutrophil function and TLR expression did not differ from healthy controls or within the patient groups. Gut permeability of MetS patients was increased significantly compared with healthy controls. L. casei Shirota administration in the MetS patients did not have any influence on any parameter tested possibly due to too-short study duration or underdosing of L. casei Shirota.

Sa2050 Increased Gut Permeability in Patients With Metabolic Syndrome

Introduction: Obesity and metabolic disorders are linked to inflammation via gut flora and/ or gut permeability. Gut derived endotoxin triggers inflammation leading to metabolic syndrome (MetS) and contributing to oxidative stress. Aim: To investigate the effect of Lactobacillus casei Shirota on gut permeability, presence of endotoxin and neutrophil function in MetS. Patients and Methods: Patients with MetS were randomized to receive 3 x 6.5x109 CFU Lactobacillus casei Shirota (probiotic group) or not for 3 months. Gut permeability was assessed by a differential sugar absorption method, endotoxin by an adapted limulus amoebocyte lysate assay, neutrophil function and toll-like receptor (TLR) expression by flow cytometry and ELISA was used to detect lipopolysaccharide binding protein (LBP) and sCD14 levels. Results: Twenty-eight patients and 10 healthy controls were included. Gut permeability was significantly increased in MetS compared to controls but did not differ between patient groups. None of the patients were positive for endotoxin. LBP and sCD14 levels were not significantly different from healthy controls. C-reactive protein and LBP levels slightly but significantly increased after 3 months within the probiotics group. Neutrophil function and TLR expression did not differ from healthy controls or within the patient groups. Discussion: Gut permeability of MetS patients was increased before markers of low grade inflammation were measurable, most likely because only a minority of patients had morbid obesity normally leading to inflammation. Lactobacillus casei Shirota did not have any influence on any parameter tested possibly due to too short study duration or underdosing of Lactobacillus casei Shirota.

Sa2052 Putative IBD Prophylaxis in Addition to Metabolic Syndrome by Oligosaccharide Synthesize Enzyme

Introduction: Obesity and metabolic disorders are linked to inflammation via gut flora and/ or gut permeability. Gut derived endotoxin triggers inflammation leading to metabolic syndrome (MetS) and contributing to oxidative stress. Aim: To investigate the effect of Lactobacillus casei Shirota on gut permeability, presence of endotoxin and neutrophil function in MetS. Patients and Methods: Patients with MetS were randomized to receive 3 x 6.5x109 CFU Lactobacillus casei Shirota (probiotic group) or not for 3 months. Gut permeability was assessed by a differential sugar absorption method, endotoxin by an adapted limulus amoebocyte lysate assay, neutrophil function and toll-like receptor (TLR) expression by flow cytometry and ELISA was used to detect lipopolysaccharide binding protein (LBP) and sCD14 levels. Results: Twenty-eight patients and 10 healthy controls were included. Gut permeability was significantly increased in MetS compared to controls but did not differ between patient groups. None of the patients were positive for endotoxin. LBP and sCD14 levels were not significantly different from healthy controls. C-reactive protein and LBP levels slightly but significantly increased after 3 months within the probiotics group. Neutrophil function and TLR expression did not differ from healthy controls or within the patient groups. Discussion: Gut permeability of MetS patients was increased before markers of low grade inflammation were measurable, most likely because only a minority of patients had morbid obesity normally leading to inflammation. Lactobacillus casei Shirota did not have any influence on any parameter tested possibly due to too short study duration or underdosing of Lactobacillus casei Shirota.

Sa2051 Safety and Tolerability of a 2-Month Course of Lactobacillus Reuteri and Effect on Circulating and Fecal Biomarkers in Healthy Adults

Introduction: Obesity and metabolic disorders are linked to inflammation via gut flora and/ or gut permeability. Gut derived endotoxin triggers inflammation leading to metabolic syndrome (MetS) and contributing to oxidative stress. Aim: To investigate the effect of Lactobacillus casei Shirota on gut permeability, presence of endotoxin and neutrophil function in MetS. Patients and Methods: Patients with MetS were randomized to receive 3 x 6.5x109 CFU Lactobacillus casei Shirota (probiotic group) or not for 3 months. Gut permeability was assessed by a differential sugar absorption method, endotoxin by an adapted limulus amoebocyte lysate assay, neutrophil function and toll-like receptor (TLR) expression by flow cytometry and ELISA was used to detect lipopolysaccharide binding protein (LBP) and sCD14 levels. Results: Twenty-eight patients and 10 healthy controls were included. Gut permeability was significantly increased in MetS compared to controls but did not differ between patient groups. None of the patients were positive for endotoxin. LBP and sCD14 levels were not significantly different from healthy controls. C-reactive protein and LBP levels slightly but significantly increased after 3 months within the probiotics group. Neutrophil function and TLR expression did not differ from healthy controls or within the patient groups. Discussion: Gut permeability of MetS patients was increased before markers of low grade inflammation were measurable, most likely because only a minority of patients had morbid obesity normally leading to inflammation. Lactobacillus casei Shirota did not have any influence on any parameter tested possibly due to too short study duration or underdosing of Lactobacillus casei Shirota.

Harnessing aptamers for electrochemical detection of endotoxin

Lipopolysaccharide (LPS), also known as endotoxin, triggers a fatal septic shock; therefore, fast and accurate detection of LPS from a complex milieu is of primary importance. Several LPS affinity binders have been reported so far but few of them have proved their efficacy in developing electrochemical sensors capable of selectively detecting LPS from crude biological liquors. In this study, we identified 10 different single-stranded DNA aptamers showing specific affinity to LPS with dissociation constants (Kd) in the nanomolar range using a NECEEM-based non-SELEX method. Based on the sequence and secondary structure analysis of the LPS binding aptamers, an aptamer exhibiting the highest affinity to LPS (i.e., B2) was selected to construct an impedance biosensor on a gold surface. The developed electrochemical aptasensor showed excellent sensitivity and specificity in the linear detection range from 0.01 to 1ng/mL of LPS with significantly reduced detection time compared with the traditional Limulus amoebocyte lysate (LAL) assay.