Plasma Endotoxin Research Articles

Role of Plasma Cytokines and Endotoxin in Patients with Acute Aortic Dissection

Objective: To analyze the role of plasma cytokines and endotoxin in patients with acute aortic dissection (AAD) in concluding prognostic effect. Material and methods: A total of 85 patients with acute aortic dissection were admitted in 2nd affiliated hospital of Zhengzhou university from January 2009 to November 2015. Patients were divided into death group and survival group. At the time of admission (T1), 12 hours after admission (T2), 24 hours after admission (T3), the plasma level of interleukin-6 (IL-6), interleukin- 10 (IL-10), tumor necrosing factor- alpha(TNF-α) and endotoxins were measured. Results: The plasma level of IL-6, TNF-α and endotoxins in death group at each time point were significantly higher than survival group (F group=3.194, 5.973, 9.156, P 0.05. Conclusion: Plasma level of IL-6, TNF-α and endotoxin are increased in the acute aortic dissections showing the progressive development of AAD. Our findings in this study indicate the role of inflammation during AAD. The changing pattern of these markers can be used for diagnosis and prophylactic treatment of AAD.

Fish Oil Reduces Hepatic Injury by Maintaining Normal Intestinal Permeability and Microbiota in Chronic Ethanol-Fed Rats.

The aim of this study was to investigate the ameliorative effects of fish oil on hepatic injury in ethanol-fed rats based on the intestinal permeability and microbiota. Rats were assigned to 6 groups and fed either a control diet or an ethanol diet such as C (control), CF25 (control with 25% fish oil), CF57 (control with 57% fish oil), E (ethanol), EF25 (ethanol with 25% fish oil), and EF57 (ethanol with 57% fish oil) groups. Rats were sacrificed at the end of 8 weeks. Plasma aspartate aminotransferase (AST) and aminotransferase (ALT) activities, hepatic cytokines, and plasma endotoxin levels were significantly higher in the E group. In addition, hepatic histopathological analysis scores in the E group were significantly elevated. Rats in the E group also showed increased intestinal permeability and decreased numbers of fecal Bifidobacterium. However, plasma AST and ALT activities and hepatic cytokine levels were significantly lower in the EF25 and EF57 groups. Histological changes and intestinal permeability were also improved in the EF25 and EF57 groups. The fecal Escherichia coli numbers were significantly lower, but fecal Bifidobacterium numbers were significantly higher in the EF25 and EF57 groups.

Endotoxin and Other Microbial Translocation Markers in the Blood: A Clue to Understand Leaky Gut Syndrome

Gut affects various systems in the human body. The leaky gut hypothesis tells us that gut microbial products cause systemic low-grade inflammation, which enhances the progression of various human diseases. Microbial translocation attributable to intestinal barrier dysfunction and hyperpermeability have been proven in major human diseases. Among these microbial products, endotoxin/ lipopolysaccharide is most extensively studied in clinical situations. However, its detection in the blood and its impact in the clinical course still arouse much discussion. Overviewing the long-standing controversies in the assay system and the main results in various clinical situations, this review regards plasma endotoxin level as a feasible microbial translocation marker. Although the detection of bacterial DNA in the blood has been gradually accepted among other microbial products, uniformity of analytical methods and usefulness in the clinical site should be established. The results on peptidoglycan, flagellin, lipoteichoic acid, and (1→ 3)-β-D-glucan in the blood are still scarce. The analysis of Toll-like receptors has suggested that several microbial products act concomitantly as pathogen-associated molecular patterns (PAMPs) in the progression of various diseases. Collective efforts to read the whole story on leaky gut and its sequences from the side of circulating microbial products may lead future progress both in patient’s care and preventive medicine.

Plasma endotoxin activity in kangaroos with oral necrobacillosis (lumpy jaw disease) using an automated handheld testing system.

The aim of the present study was to evaluate the reliability and effectiveness of directly determining endotoxin activity in plasma samples from kangaroos with lumpy jaw disease (LJD, n=15) and healthy controls (n=12). Prior to the present study, the ability of the commercially available automated handheld portable test system (PTSTM) to detect endotoxin activity in kangaroo plasma was compared with that of the traditional LAL-kinetic turbidimetric (KT) assay. Plasma samples, which were obtained from endotoxin-challenged cattle, were diluted 1:20 in endotoxin-free water and heated to 80°C for 10 min. The performance of the PTSTM was not significantly different from that of the traditional LAL-based assay. The data obtained using PTSTM correlated with those using KT (r2=0.963, P<0.001). These findings indicated that the PTSTM is applicable as a simplified system to assess endotoxin activity in macropods. In the present study, we demonstrated the diagnostic value of plasma endotoxin activity in kangaroos with systemic inflammation caused by oral necrobacillosis and identified plasma endotoxin activity as a sensitive marker of systemic inflammation in kangaroos with LJD. Based on ROC curves, we proposed a diagnostic cut-off point for endotoxin activity of >0.22 EU/ml for the identification of LJD. Our results indicate that the assessment of plasma endotoxin activity is a promising diagnostic tool for determining the outcome of LJD in captive macropods.

Inflammation Related MicroRNAs Are Modulated in Total Plasma and in Extracellular Vesicles from Rats with Chronic Ingestion of Sucrose.

Circulating microRNAs (miRNAs) and the functional implications of miRNAs contained in extracellular vesicles (EVs) have gained attention in the last decade. Little is known about the regulation of the abundance of plasma miRNAs in response to chronic ingestion of carbohydrates. Therefore, we explored the circulating levels of miR-21, miR-146a, miR-155, and miR-223 in rats consuming sucrose in drinking water. Weanling Wistar rats were 25 weeks with 30% sucrose in drinking water, and miRNAs expression was determined in total plasma and in microvesicles, by RT-qPCR with TaqMan probe based assays for miR-21, miR-146a, miR-155, and miR-223, using cel-miR-39 (as spike in control and reference). Endotoxemia was also measured. Sucrose-fed animals showed higher body weight and retroperitoneal adipose tissue as well as higher glucose and triglyceride plasma levels than controls. Plasma endotoxin levels were low and not different among groups. Plasma miR-21 and miR-223 were higher in the sucrose group (p < 0.05), whereas miR-155 tended to be lower (p = 0.0661), and miR-146a did not show significant differences. In the plasma EVs the same trend was found except for miR-146a that showed significantly higher levels (p < 0.05). Overall, our results show that high carbohydrate ingestion modulates circulating miRNAs levels related to an inflammatory response.

Propofol increases morbidity and mortality in a rat model of sepsis.

IntroductionSevere sepsis is associated with approximately 50% mortality and accounts for tremendous healthcare costs. Most patients require ventilatory support and propofol is commonly used to sedate mechanically ventilated patients. Volatile anesthetics have been shown to attenuate inflammation in a variety of different settings. We therefore hypothesized that volatile anesthetic agents may offer beneficial immunomodulatory effects during the course of long-term intra-abdominal sepsis in rats under continuous sedation and ventilation for up to 24 hours.MethodsSham operation or cecal ligation and puncture (CLP) was performed in adult male Wistar rats followed by mechanical ventilation. Animals were sedated for 24 hours with propofol (7 to 20 mg/kg/h), sevoflurane, desflurane or isoflurane (0.7 minimal alveolar concentration each).ResultsSeptic animals sedated with propofol showed a mean survival time of 12 hours, whereas >56% of all animals in the volatile groups survived 24 hours (P <0.001). After 18 hours, base excess in propofol + CLP animals (−20.6 ± 2.0) was lower than in the volatile groups (isoflurane + CLP: -11.7 ± 4.2, sevoflurane + CLP: -11.8 ± 3.5, desflurane + CLP -14.2 ± 3.7; all P <0.03). Plasma endotoxin levels reached 2-fold higher levels in propofol + CLP compared to isoflurane + CLP animals at 12 hours (P <0.001). Also blood levels of inflammatory mediators (tumor necrosis factor-α, interleukin-1β, interleukin-10, CXCL-2, interferon-γ and high mobility group protein-1) were accentuated in propofol + CLP rats compared to the isoflurane + CLP group at the same time point (P <0.04).ConclusionsThis is the first study to assess prolonged effects of sepsis and long-term application of volatile sedatives compared to propofol on survival, cardiovascular, inflammatory and end organ parameters. Results indicate that volatile anesthetics dramatically improved survival and attenuate systemic inflammation as compared to propofol. The main mechanism responsible for adverse propofol effects could be an enhanced plasma endotoxin concentration, leading to profound hypotension, which was unresponsive to fluid resuscitation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13054-015-0751-x) contains supplementary material, which is available to authorized users.

Preventing Gut Leakiness and Endotoxemia Contributes to the Protective Effect of Zinc on Alcohol-Induced Steatohepatitis in Rats1–3

Background: Zinc deficiency has been well documented in alcoholic liver disease.Objective: This study was undertaken to determine whether dietary zinc supplementation provides beneficial effects in treating alcohol-induced gut leakiness and endotoxemia.Methods: Male Sprague Dawley rats were divided into 3 groups and pair-fed (PF) Lieber-DeCarli liquid diet for 8 wk: 1) control (PF); 2) alcohol-fed (AF; 5.00–5.42% wt:vol ethanol); and 3) AF with zinc supplementation (AF/Zn) at 220 ppm zinc sulfate heptahydrate. The PF and AF/Zn groups were pair-fed with the AF group. Hepatic inflammation and endotoxin signaling were determined by immunofluorescence and quantitative polymerase chain reaction (qPCR). Alterations in intestinal tight junctions and aldehyde dehydrogenases were assessed by qPCR and Western blot analysis.Results: The AF rats had greater macrophage activation and cytokine production (P < 0.05) in the liver compared with the PF rats, whereas the AF/Zn rats showed no significant differences (P > 0.05). Plasma endotoxin concentrations of the AF rats were 136% greater than those of the PF rats, whereas the AF/Zn rats did not differ from the PF rats. Ileal permeability was 255% greater in the AF rats and 19% greater in the AF/Zn rats than in the PF rats. The AF group had reduced intestinal claudin-1, occludin, and zona occludens-1 (ZO-1) expression, and the AF/Zn group had upregulated claudin-1 and ZO-1 expression (P < 0.05) compared with the PF group. The intestinal epithelial expression and activity of aldehyde dehydrogenases were elevated (P < 0.05) in the AF/Zn rats compared with those of the AF rats. Furthermore, the ileal expression and function of hepatocyte nuclear factor 4α, which was impaired in the AF group, was significantly elevated in the AF/Zn group compared with the PF group.Conclusions: The results demonstrate that attenuating hepatic endotoxin signaling by preserving the intestinal barrier contributes to the protective effect of zinc on alcohol-induced steatohepatitis in rats.

Modulation of Intestinal Barrier and Bacterial Endotoxin Production Contributes to the Beneficial Effect of Nicotinic Acid on Alcohol-Induced Endotoxemia and Hepatic Inflammation in Rats.

Alcohol consumption causes nicotinic acid deficiency. The present study was undertaken to determine whether dietary nicotinic acid supplementation provides beneficial effects on alcohol-induced endotoxin signaling and the possible mechanisms at the gut-liver axis. Male Sprague-Dawley rats were pair-fed the Lieber-DeCarli liquid diets containing ethanol or isocaloric maltose dextrin for eight weeks, with or without dietary supplementation with 750 mg/liter nicotinic acid. Chronic alcohol feeding elevated the plasma endotoxin level and activated hepatic endotoxin signaling cascade, which were attenuated by nicotinic acid supplementation. Alcohol consumption remarkably decreased the mRNA levels of claudin-1, claudin-5, and ZO-1 in the distal intestine, whereas nicotinic acid significantly up-regulated these genes. The concentrations of endotoxin, ethanol, and acetaldehyde in the intestinal contents were increased by alcohol exposure, and niacin supplementation reduced the intestinal endotoxin and acetaldehyde levels. Nicotinic acid supplementation upregulated the intestinal genes involved in aldehyde detoxification via transcriptional regulation. These results demonstrate that modulation of the intestinal barrier function and bacterial endotoxin production accounts for the inhibitory effects of nicotinic acid on alcohol-induced endotoxemia and hepatic inflammation.

EICOSAPENTAENOIC ACID ENHANCES HEATSTROKE-IMPAIRED INTESTINAL EPITHELIAL BARRIER FUNCTION IN RATS.

Dysfunction of the intestinal barrier plays an important role in the pathological process of heatstroke. Omega-3 (or n-3) polyunsaturated fatty acids, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), help protect the intestinal mucosal barrier. This study assessed if pretreating rats with EPA or DHA could alleviate heat stress-induced damage to the intestinal barrier caused by experimental heatstroke. Male Wistar rats were pregavaged with either EPA, DHA, corn oil, or normal saline (all 1 g/kg) for 21 days before the heatstroke experiment (control rats were not exposed to heat). Experimental rats were exposed to an ambient temperature of 37°C and 60% humidity to induce heatstroke, and then they were allowed to recover at room temperature after rapid cooling. Survival time of rats was monitored after heatstroke. Horseradish peroxidase flux from the gut lumen and the level of plasma D-lactate were measured to analyze intestinal permeability at 6 h after heatstroke. Plasma endotoxin levels were determined using a limulus amoebocyte lysate assay. Expressions of the tight junction (TJ) proteins occludin and ZO-1 were analyzed by Western blot and localized by immunofluorescence microscopy. Tight junction protein morphology was observed by transmission electron microscopy. Fatty acids of ileal mucosa were analyzed using gas chromatography-mass selective detector. Eicosapentaenoic acid significantly increased survival time after heatstroke. Eicosapentaenoic acid significantly decreased intestinal permeability and plasma endotoxin levels. Eicosapentaenoic acid effectively attenuated the heatstroke-induced disruption of the intestinal structure and improved the histology score, whereas DHA was less effective, and corn oil was ineffective. Pretreatment with EPA also increased expression of occludin and ZO-1 to effectively prevent TJ disruption. Eicosapentaenoic acid pretreatment enriched itself in the membrane of intestinal cells. Our results indicate that EPA pretreatment is more effective than DHA pretreatment in attenuating heat-induced intestinal dysfunction and preventing TJ damage. Enhanced expression of TJ proteins that support the epithelial barrier integrity may be important for maintaining a functional intestinal barrier during heatstroke.

Dysbiosis of Intestinal Microbiota Associated With Inflammation Involved in the Progression of Acute Pancreatitis.

To evaluate alterations of the intestinal bacteria and its associations with the inflammation in acute pancreatitis (AP). A multihospital prospective clinical study was conducted, and a total of 108 participants were enrolled in our study, including 44 with severe AP (SAP), 32 with mild AP (MAP), and 32 healthy volunteers. The structure of intestinal microbiota, 10 predominant bacteria, plasma endotoxin, and serum cytokines were investigated by polymerase chain reaction-denaturing gradient gel electrophoresis, real-time quantitative polymerase chain reaction, Limulus amebocyte lysate tests, and enzyme-linked immunosorbent assays, respectively. Dramatic alterations in the predominant fecal microbiota were observed in most of both MAP and SAP patients. In addition, the rates of the multiorgan failures and infectious complications in the patients with SAP with altered intestinal microbiota were significantly higher than in those whose intestinal microbiota remained unaltered. Enterococcus increased and Bifidobacterium decreased in the patients with SAP compared to the patients with MAP. Serum IL-6 were positively correlated with Enterobacteriaceae and Enterococcus and negatively correlated with Bifidobacterium, whereas plasma endotoxin positively correlated with Enterococcus (P < 0.05). The intestinal bacteria most frequently altered in both the patients with MAP and those with SAP significantly correlated with inflammation, which indicated that the intestinal microbiota may be involved in the progression of AP.

Associations of Soluble CD14 and Endotoxin with Mortality, Cardiovascular Disease, and Progression of Kidney Disease among Patients with CKD.

CD14 plays a key role in the innate immunity as pattern-recognition receptor of endotoxin. Higher levels of soluble CD14 (sCD14) are associated with overall mortality in hemodialysis patients. The influence of kidney function on plasma sCD14 levels and its relationship with adverse outcomes in patients with CKD not yet on dialysis is unknown. This study examines the associations between plasma levels of sCD14 and endotoxin with adverse outcomes in patients with CKD. We measured plasma levels of sCD14 and endotoxin in 495 Leuven Mild-to-Moderate CKD Study participants. Mild-to-moderate CKD was defined as presence of kidney damage or eGFR<60 ml/min per 1.73 m(2) for ≥3 months, with exclusion of patients on RRT. Study participants were enrolled between November 2005 and September 2006. Plasma sCD14 was negatively associated with eGFR (ρ=-0.34, P<0.001). During a median follow-up of 54 (interquartile range, 23-58) months, 53 patients died. Plasma sCD14 was predictive of mortality, even after adjustment for renal function, Framingham risk factors, markers of mineral bone metabolism, and nutritional and inflammatory parameters (hazard ratio [HR] per SD higher of 1.90; 95% confidence interval [95% CI],1.32 to 2.74; P<0.001). After adjustment for the same risk factors, plasma sCD14 was also a predictor of cardiovascular disease (HR, 1.30; 95% CI, 1.00 to 1.69; P=0.05). Although plasma sCD14 was associated with progression of CKD, defined as reaching ESRD or doubling of serum creatinine in models adjusted for CKD-specific risk factors (HR, 1.24; 95% CI, 1.01 to 1.52; P=0.04), significance was lost when adjusted for proteinuria (HR, 1.19; 95% CI, 0.96 to 1.48; P=0.11). There was neither correlation between plasma endotoxin and sCD14 (ρ=-0.06, P=0.20) nor was endotoxin independently associated with adverse outcome during follow-up. Plasma sCD14 is elevated in patients with decreased kidney function and associated with mortality and cardiovascular disease in patients with CKD not yet on dialysis.

Effects of dietary essential oil and enzyme supplementation on growth performance and gut health of broilers challenged by Clostridium perfringens

Abstract An experiment was carried out to evaluate the effects of dietary supplemental essential oil comprising thymol and carvacrol (EO), and enzyme complex containing xylanase, glucanase and mannanase as major components (CSM) on Clostridium perfringens challenged model in broilers. A total of 648 1-d-old female broiler chicks were assigned to eight groups. A 2 × 2 × 2 factorial arrangement of treatments was used in a randomized complete block design to study the effects of essential oil supplementation (with or without EO 60 mg/kg diet), enzymes addition (with or without CSM 500 mg/kg diet), pathogen challenge (with or without C. perfringens challenge from d 14 to 20), and the interactive effects. A pelleted high NSP wheat–soybean meal basal diet was formulated to meet nutrient requirements recommended by NRC (1994) . Body weight, feed intake, and feed to gain ratio were determined on d 14, 21 and 35; ileal digesta viscosity, intestinal lesion score, ileal histomorphologic indices, bacterial population and plasma endotoxin were measured on d 21; while ileal permeability was determined from d 24 to 26. Results indicated that enzyme CSM supplementation increased body weight on d 21 and 35, improved feed to gain ratio from d 0 to 14 and 0 to 21, increased P-glycoprotein dependent efflux permeability ( P app (S–M)), decreased digesta viscosity, and lowered ileal C. perfringens and Lactobacillus colonization. CSM supplementation could also improve feed conversion ratio from d 0 to 35 and VH/CD on d 21 of C. perfringens challenged chicks, while EO supplementation increased villus height and VH/CD. C. perfringens challenge increased intestinal lesion score, C. perfringens and Lactobacillus colonization, but decreased Bifidobacterium colonization and P app (S–M). C. perfringens challenge also resulted in increased plasma endotoxin, passive transcellular permeability ( P app (M–S)), sIgA, and E. coli colonization when chicks were fed with basal diet, while EO or CSM supplementation could alleviate the alteration caused by C. perfringens infection. In conclusion, dietary enzyme CSM supplementation could benefit for growth performance and gut health, and EO could improve the gut health of C. perfringens challenged chicks.

Plasma endotoxin core antibody concentration and linear growth are unrelated in rural Malawian children aged 2-5 years.

BackgroundEnvironmental enteropathy is subclinical inflammation of the upper gastrointestinal tract associated with reduced linear growth in developing countries. Usually investigators have used biopsy or a dual sugar absorption test to assess environmental enteropathy. Such tests are time and resource intensive, restricting their utility as screening methods. Serum endotoxin core antibody (EndoCab) concentration is a potential indicator of intestinal inflammation and integrity, and thus may be useful to predict environmental enteropathy. We analyzed the association of serum EndoCab levels versus linear growth and lactulose-mannitol assay results in 2–5 year old rural Malawian children.MethodsThis was an observational study of 388 rural, asymptomatic Malawian children who had anthropometric measurements taken at least every 3 months since birth. In June and July 2011, dual sugar permeability tests were performed and serum samples were drawn for EndoCab assays. Pearson correlation, Student’s t test and multivariable linear regression were used to compare ln EndoCab concentrations with height-for-age z scores (HAZ) at time of sampling and 3 months later. Identical analysis was also performed for ln EndoCab versus measurements from dual sugar permeability testing performed in conjunction with serum sampling. In a subgroup of children with anthropometric data in the months prior to serum sampling, Pearson correlation was used to estimate the relationship between ln EndoCab and recent linear growth.ResultsLn EndoCab concentrations were not correlated with HAZ at time of measurement (B = −0.078, P = 0.14) nor change in HAZ over the subsequent 3 months HAZ (B = −0.018, P = 0.27). EndoCab concentration was not associated with %lactulose excretion (B < 0.001, P = 0.98) nor the lactulose:mannitol ratio (B = 0.021, P = 0.62). Subgroup analysis also did not reveal any significant association between EndoCab and recent growth.ConclusionEndoCab titers were not correlated with measurements of growth or intestinal permeability in rural pre-school aged Malawian children.

PWE-088 Frequency and function of anti-bacterial mait cells are significantly impaired in advanced alcoholic liver disease

Introduction Intestinal bacterial translocation and systemic gut-derived bacterial products play a central role in the immunopathogenesis of alcoholic liver disease (ALD), yet the mechanisms of susceptibility to infection and the link with intestinal immunity remain elusive. Mucosal associated invariant T cells (MAIT) are unconventional T cells which only respond to bacteria-derived metabolites and are found in large numbers in the blood, intestinal mucosae and liver. They represent a key sentinel system for the homeostatic control of the gut flora and for the control of bacterial infections. The aim of this study was to assess the role of MAIT cells in ALD. Method Peripheral blood mononuclear cells from subjects with acute alcoholic hepatitis (AAH, Maddrey’s discriminant function >32; n = 9), compensated alcohol-related cirrhosis (ARC, n = 9) and healthy controls (HC, n = 9) were examined by FACS. MAIT cells were identified as CD161 + /Vα7.2+ CD8 + T cells and MAIT-presenting cells (MPC) as MR1+ monocytes or B cells. We quantified (1) frequency, activation status (CD69/HLA-DR) and immunoinhibitory signatures (PD1/TIM3/LAG3) of MAIT cells; (2) frequency and immunoinhibitory status (PD1/PDL1/TIM3/mGal9) of MPC; (3) cytokine/cytotoxicity profiles (IFNγ/TNFα/IL17; GranzymeB (GrB)/Perforin/CD107a) of MAIT cells after in-vitro stimulation with fixed E. Coli. Plasma cytokines and endotoxin levels were assessed by ELISA. Results MAIT frequencies were reduced in ARC (p = 0.005) and dramatically lower in AAH (p Conclusion This is the first report addressing the role of MAIT cells in liver disease. We show that MAIT cells have quantitative and functional impairments in ALD, with defective TNFα and IL17 responses, increased killing potential and overexpression of immunoinhibitory receptors. MAIT cells may represent a novel immunotherapeutic target for ALD. Disclosure of interest None Declared.

760 Confocal Endomicroscopy Demonstrates Increased Intestinal Permeability and Gut Dysfunction in Patients With Acute and Chronic Pancreatitis

Introduction Increased intestinal permeability and endotoxaemia occur in acute pancreatitis (AP) but have yet to be confirmed in chronic pancreatitis (CP). Confocal endomicroscopy (CEM) is a novel technology that has identified epithelial gaps and leakage of intravenously administered fluorescein from small intestinal mucosa in inflammatory bowel diseases. We aimed to determine intestinal permeability changes in AP and CP using CEM, assessing associations with endotoxaemia, circulating cytokines and alterations in duodenal flora. Method 182 patients were recruited: 35 mild AP; 11 moderate AP; 8 severe AP (Determinant Based Classification); 47 CP (27 surgically managed, 20 conservatively managed); 33 chronic liver disease and 48 non-ulcer dyspepsia (from whom 14 healthy H. pylorinegative controls were identified). Blood was sampled and duodenal fluid aspirated for culture. CEM was performed in consenting patients and fluorescein leakage scored by two independent blinded investigators. Plasma endotoxin antibodies, serum cytokine concentrations and lactulose:mannitol ratios were measured; D-amino oxidase concentrations were assayed in AP patients undergoing CEM. Results Fluorescein leakage from epithelial villi at CEM and lactulose:mannitol ratios were significantly higher in AP patients (p = 0.0346 and p = 0.046 respectively) and surgically managed CP (p = 0.032, p = 0.030) compared to healthy controls. Patients with AP more frequently showed positive duodenal bacterial cultures than controls (p = 0.004); positive duodenal cultures were associated with fluorescein leakage (p = 0.035). Plasma IgM and IgG concentrations were decreased in AP compared to controls, most markedly in severe AP, and multiple cytokines showed significantly increased concentrations in severe AP. D-amino oxidase enzyme concentrations were significantly increased in those AP patients who showed fluorescein leakage at CEM (p = 0.035). Conclusion CEM identified increased intestinal permeability in AP and surgically managed CP, associated with endotoxaemia, increased serum inflammatory cytokines and D-amino oxidase. These changes were most marked in severe AP. The association of fluorescein leakage with duodenal bacterial proliferation in AP indicates major gut dysfunction that may account for bacterial translocation via epithelial gaps, contributing to AP severity. Disclosure of interest None Declared.

Mo1824 miR-511-3p, Embedded in the Macrophage Mannose Receptor Gene, Contributes to Experimental Colitis

Approximately 40% of inflammatory bowel diseases (IBD) are associated with thromboembolic events causing significant rate of morbidity and mortality. These extra intestinal events of IBD are initiated by the qualitative and quantitative abnormalities in both coagulation and fibrinolytic factors producing states of hypercoagulability. Nevertheless, the interrelationship between IBD pathogenesis and hypercoagulability condition is not well understood (Figure 1). Here we examine the role of a coagulable state triggered by impaired fibrinolysis in the development of chronic colonic inflammation. Plasminogen (Plg) is the primary fibrinolytic enzyme, becomes activated to serine protease plasmin to dissolve the fibrin clot and to counterbalance hypercoagulability. The rapidity of IBD is caused by combining the effects of genetic and environmental factors. Our data show that when fed with a high fat diet (HFD, 45% milk fat), mouse knockout of Plg (Plg-/mice) developed continuous lesions in the colon with multiple inflammatory polyps (as of 8 weeks, n=15) bearing ulcers. This pathophysiology was not observed in HFD fed wild type (Plg+/+) mice and standard chow diet fed Plg+/+ and Plg-/mice. The increased inflammatory cell infiltration in the colon of HFD fed Plg-/mice was corroborated with increased levels of pro-inflammatory cytokines (IL-6, IL-17 & TNF alpha). In addition, HFD-fed Plg-/mice expressed reduced levels of colonic epithelial tight junctions (Claudin1, Occludin and ZO1), a finding compatible with an impaired epithelial barrier function, compared to Plg+/+ mice. Moreover, HFD fed Plg-/mice showed increased levels of plasma endotoxin, an indicator of impaired intestinal barrier function, compared with HFD-fed Plg+/+ mice. All these pathologies of HFD fed Plg-/mice were associated with excessive fibrin deposition in both vascular and extravascular space in the colon compared with HFD fed Plg+/+ mice providing a link between fibrin deposition and inflammation. Altogether, our present data imply that fibrinolytic pathway plays a crucial role in colon homeostasis and defects in this pathway can cause impaired colon barrier function and sustained colonic inflammation. In depth exploration of this pathway will provide a clue towards the potential role of Plg in colon homeostasis. This study will also challenge current paradigms on the relationship between coagulation abnormalities and IBD, which presume that these abnormalities are a consequence rather than a cause of the disease.

Neutrophilia Delays Equine Neutrophil Apoptosis

Regulation of neutrophil apoptosis has assumed a critical role in inflammation. The objective of this study was to determine the effect of neutrophil concentration on their apoptosis in vitro and ex vivo in horses with experimental intestinal ischemia and reperfusion injury. Neutrophils were isolated from healthy horses randomized to the following treatments: sham celiotomy (CEL, n=4), intestinal ischemia and reperfusion (IR, n=6), intestinal ischemia and reperfusion with gadolinium chloride treatment to deplete pulmonary intravascular macrophages (IRGC, n=6). All horses experienced a significant neutrophilia following surgery without detectable plasma endotoxin concentrations (&lt;0.06 EU/ml). Isolated neutrophils were cultured for 12 or 24 hours at concentrations of 2, 5, or 8 x 106 cells/ml and evaluated for the occurrence of apoptosis using Annexin V and propidium iodide staining. Caspase‐3, ‐8, and ‐9 activities were measured in cell lysates before and after treatment. Delayed neutrophil apoptosis was highly correlated (12 h incubation: r = ‐0.75, 24 h incubation: r = ‐0.57) with increasing neutrophil concentration in vitro. Neutrophil apoptosis was significantly delayed ex vivo after treatment, except in IRGC horses (12 h incubation: CEL: P =0.03, IR: P = 0.05;24 h incubation: CEL:P = 0.001, IR: P = 0.004). Caspase‐3, ‐8, and ‐9 activities were reduced in neutrophils isolated after surgery in all groups. The data show that equine neutrophil apoptosis is delayed in vitro and ex vivo via down‐regulation of caspase activity when neutrophils become more concentrated, which could have implications for the development and progression of systemic inflammatory responses.

Mo1825 The Interaction of Enteric Bacterial Effectors With Host Engulfment Pathway in Phagocytes Regulates the Pathogenesis of Infection

Approximately 40% of inflammatory bowel diseases (IBD) are associated with thromboembolic events causing significant rate of morbidity and mortality. These extra intestinal events of IBD are initiated by the qualitative and quantitative abnormalities in both coagulation and fibrinolytic factors producing states of hypercoagulability. Nevertheless, the interrelationship between IBD pathogenesis and hypercoagulability condition is not well understood (Figure 1). Here we examine the role of a coagulable state triggered by impaired fibrinolysis in the development of chronic colonic inflammation. Plasminogen (Plg) is the primary fibrinolytic enzyme, becomes activated to serine protease plasmin to dissolve the fibrin clot and to counterbalance hypercoagulability. The rapidity of IBD is caused by combining the effects of genetic and environmental factors. Our data show that when fed with a high fat diet (HFD, 45% milk fat), mouse knockout of Plg (Plg-/mice) developed continuous lesions in the colon with multiple inflammatory polyps (as of 8 weeks, n=15) bearing ulcers. This pathophysiology was not observed in HFD fed wild type (Plg+/+) mice and standard chow diet fed Plg+/+ and Plg-/mice. The increased inflammatory cell infiltration in the colon of HFD fed Plg-/mice was corroborated with increased levels of pro-inflammatory cytokines (IL-6, IL-17 & TNF alpha). In addition, HFD-fed Plg-/mice expressed reduced levels of colonic epithelial tight junctions (Claudin1, Occludin and ZO1), a finding compatible with an impaired epithelial barrier function, compared to Plg+/+ mice. Moreover, HFD fed Plg-/mice showed increased levels of plasma endotoxin, an indicator of impaired intestinal barrier function, compared with HFD-fed Plg+/+ mice. All these pathologies of HFD fed Plg-/mice were associated with excessive fibrin deposition in both vascular and extravascular space in the colon compared with HFD fed Plg+/+ mice providing a link between fibrin deposition and inflammation. Altogether, our present data imply that fibrinolytic pathway plays a crucial role in colon homeostasis and defects in this pathway can cause impaired colon barrier function and sustained colonic inflammation. In depth exploration of this pathway will provide a clue towards the potential role of Plg in colon homeostasis. This study will also challenge current paradigms on the relationship between coagulation abnormalities and IBD, which presume that these abnormalities are a consequence rather than a cause of the disease.

Kupffer cell depletion protects against the steatosis, but not the liver damage, induced by marginal-copper, high-fructose diet in male rats.

High-fructose feeding impairs copper status and leads to low copper availability, which is a novel mechanism in obesity-related fatty liver. Copper deficiency-associated hepatic iron overload likely plays an important role in fructose-induced liver injury. Excess iron in the liver is distributed throughout hepatocytes and Kupffer cells (KCs). The aim of this study was to examine the role of KCs in the pathogenesis of nonalcoholic fatty liver disease induced by a marginal-copper high-fructose diet (CuMF). Male weanling Sprague-Dawley rats were fed either a copper-adequate or a marginally copper-deficient diet for 4 wk. Deionized water or deionized water containing 30% fructose (wt/vol) was also given ad libitum. KCs were depleted by intravenous administration of gadolinium chloride (GdCl3) before and/or in the middle of the experimental period. Hepatic triglyceride accumulation was completely eliminated with KC depletion in CuMF consumption rats, which was associated with the normalization of elevated plasma monocyte chemoattractant protein-1 (MCP-1) and increased hepatic sterol regulatory element binding protein-1 expression. However, hepatic copper and iron content were not significantly affected by KC depletion. In addition, KC depletion reduced body weight and epididymal fat weight as well as adipocyte size. Plasma endotoxin and gut permeability were markedly increased in CuMF rats. Moreover, MCP-1 was robustly increased in the culture medium when isolated KCs from CuMF rats were treated with LPS. Our data suggest that KCs play a critical role in the development of hepatic steatosis induced by marginal-copper high-fructose diet.

Positive regulatory effects of perioperative probiotic treatment on postoperative liver complications after colorectal liver metastases surgery: a double-center and double-blind randomized clinical trial.

BackgroundColorectal liver metastases (CLM) occur frequently and postoperative intestinal infection is a common complication. Our previous study showed that probiotics could decrease the rate of infectious complications after colectomy for colorectal cancer. To determine the effects of the perioperative administration of probiotics on serum zonulin levels which is a marker of intestinal permeability and the subsequent impact on postoperative infectious complications in patients with CLM.Methods150 patients with CLM were randomly divided into control group (n = 68) and probiotics group (n = 66). Probiotics and placebo were given orally for 6 days preoperatively and 10 days postoperatively to control group and probiotics group respectively. We used the local resection for metastatic tumor ,while for large tumor, the segmental hepatectomy. Postoperative outcome were recorded. Furthermore, complications in patients with normal intestinal barrier function and the relation with serum zonulin were analyzed to evaluate the impact on the liver barrier dysfunction.ResultsThe incidence of infectious complications in the probiotics group was lower than control group. Analysis of CLM patients with normal postoperative intestinal barrier function paralleled with the serum zonulin level. And probiotics could also reduce the concentration of serum zonulin (P = 0.004) and plasma endotoxin (P < 0.001).ConclusionPerioperative probiotics treatment could reduce the serum zonulin level, the rate of postoperative septicemia and maintain the liver barrier in patients undergoing CLM surgery. we propose a new model about the regulation of probiotics to liver barrier via clinical regulatory pathway. We recommend the preoperative oral intake of probiotics combined with postoperative continued probiotics treatment in patients who undergo CLM surgery.Trial registrationChiCTR-TRC-12002841. 2012/12/21Electronic supplementary materialThe online version of this article (doi:10.1186/s12876-015-0260-z) contains supplementary material, which is available to authorized users.