LPS Infusion Research Articles

Effects of epinephrine on heart rate variability and cytokines in a rat sepsis model.

Catecholamines have both anti-inflammatory and vasoactive properties. A decreased cardiac response to catecholamines has been associated with a high risk of death in sepsis and septic shock. The aim of this study was to investigate the effects of epinephrine (EPI) on heart rate variability (HRV) and autonomic balance, as well as cytokine levels, in a rat sepsis model. Thirty-six male Sprague-Dawley rats were assigned to 4 experimental groups and 2 control groups of 6 rats each. The rats in the experimental groups were inoculated with a lipopolysaccharide (LPS, endotoxin) to establish a sepsis model. Group A received only LPS; group B received LPS, antecedent EPI and the nonselective β-blocker propranolol; group C received LPS and antecedent EPI; and group D received LPS, antecedent EPI and the selective β1-blocker esmolol. One control group received EPI and the other received saline placebo. Heart rate variability (HRV) was analyzed and tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) levels were measured. Measurements were carried out at baseline, at 0 hour after EPI infusion, and at 0.5, 2, and 4 hours after LPS inoculation. There were significant differences in HRV and cytokine levels between the groups, indicating that LPS infusion caused autonomic imbalance. Antecedent EPI significantly decreased the level of TNF-α in group C compared with group A in which TNF-α level peaked at 2 hours and then declined. Propranolol (group B) but not esmolol (group D) administration resulted in elevated TNF-α levels, comparable to those observed in group A. In conclusion, antecedent administration of EPI in a rat sepsis model inhibits the production of TNF-α possibly via the β2-adrenoceptor.

Transfusion of autologous extracellular vesicles from stored red blood cells does not affect coagulation in a model of human endotoxemia

Red blood cell (RBC) transfusion has been related to thromboembolic events. Microvesicles in the RBC product may support coagulation because they have procoagulant effects in vitro. We investigated whether transfusion of RBCs containing extracellular vesicles promotes coagulation in human recipients. As transfusion is mostly administered to ill patients, we used a model of endotoxemia. Eighteen healthy volunteers were randomized to receive either saline or fresh (2 days stored) or stored autologous (35 days stored) RBC transfusion (Dutch Trial Register: NTR4455). Two hours after infusion of lipopolysaccharide (LPS, from Escherichia coli, 2 ng/kg body weight), subjects received either saline or fresh or stored RBCs. Blood was sampled every 2 hours up to 8 hours after LPS infusion. Vesicles were measured with a flow cytometer (A50-Micro, Apogee Flow Systems). LPS resulted in increased thrombin generation compared to baseline. During storage, the total number of extracellular vesicles increased from 1.4 × 108 /mL (interquartile range [IQR], 8.3 × 107 -1.9 × 108 /mL) in the fresh product to 1.7 × 1010 /mL (IQR, 7.9 × 109 -2.3 × 1010 /mL; p < 0.01) in the stored product (p < 0.001). Vesicles appeared to be mostly RBC derived. After transfusion, extracellular vesicles from stored RBC products, but not from fresh products, could be detected in the circulation of healthy volunteers. However, infusion of stored RBC extracellular vesicles did not augment thrombin generation compared to endotoxemic controls. Also, levels of d-dimer and thrombin-antithrombin complex were unaffected. In conclusion, transfusion of autologous RBCs containing high levels of extracellular vesicles does not enhance coagulation in human volunteers with endotoxemia.

Lipopolysaccharide exposure modifies salivary and circulating level of cortisol in goats

The aim of this study was to investigate response of salivary and blood cortisol level and cardinal physiological variables in presence of endotoxaemia in goats. For this, eight healthy adult male Jakhrana goats (3–4 years of age; 41.9 ± 1.9 kg BW) were adapted to experimental conditions for 10 days, after which, they were randomly assigned equally into two groups i.e. control (CON; 20 μL/kg BW i.v. physiological saline) or lipopolysaccharide (LPS; 1 μg/kg BW i.v.; Escherichia coli 0111:B4). Recording of cardinal physiological variables [rectal temperature (RT), respiratory rate (RR) and heart rate (HR)] was done at about 10 min before (baseline) and at different time intervals (2, 4, 6, 8, 26, 32 and 48 h) after saline or LPS infusion. Immediately thereafter, matched saliva and blood samples were collected at similar time points to investigate time dependent changes in cortisol concentration after LPS administration. The results showed that cortisol concentration in both saliva and blood plasma were increased significantly (P < 0.05) at 2 h and remains elevated in both the biological fluids up to 8 h after LPS infusion. Correlation analysis showed a significant positive relationship of salivary cortisol with plasma cortisol concentration (r = 0.556, P < 0.001). After systemic LPS exposure, increased HR and RR were observed earlier (at 2 h after LPS infusion) compared to the increase in RT (at 4 h after LPS infusion) (P < 0.05). The results of the present study demonstrated that the measurement of salivary and blood cortisol level could be a practical tool for monitoring time dependent effects of LPS and for evaluating early inflammatory response to acute bacterial infection in goats.

In vivo lung perfusion rehabilitates sepsis-induced lung injury

BackgroundSepsis is the leading cause of lung injury in adults and can lead to acute respiratory distress syndrome (ARDS). Using a novel technique of isolated in vivo lung perfusion (IVLP), we hypothesized that normothermic IVLP will improve oxygenation and compliance in a porcine model of sepsis-induced lung injury. MethodsMature adult swine (n = 8) were administered lipopolysaccharide (LPS; 50 μg/kg over 2 hours) via the external jugular vein, followed by sternotomy and central extracorporeal membrane oxygenation (ECMO) cannulation (right atrium to ascending aorta). The left pulmonary artery (inflow) and left superior and inferior pulmonary veins (outflow) were dissected out and cannulated to deliver isolated perfusion to the left lung. After 4 hours of normothermic IVLP with Steen solution, the left lung then underwent 4 hours of reperfusion after IVLP decannulation. Airway pressures and lung-specific pulmonary vein blood gases from the right lung (LPS control) and left lung (LPS + IVLP) of the same animal were compared. ResultsAll animals demonstrated a significant reduction in the ratio of partial pressure of oxygen in arterial blood (PaO2)/fraction of inspired oxygen (FiO2) (P/F ratio) and total lung compliance at 2 hours after the start of LPS infusion (mean, 469 ± 19.7 mm Hg vs 222.2 ± 21.4 mm Hg; P < .0001). After reperfusion, 6 animals (75%) exhibited improved lung function, allowing for ECMO decannulation. Lung-specific oxygenation was superior in the left lung after 4 hours of reperfusion (mean, 310.5 ± 54.7 mm Hg vs 201.1 ± 21.7 mm Hg; P = .01). Similarly, total lung compliance improved after IVLP of the left lung. The lung wet weight to dry weight ratio demonstrated reduced edema in rehabilitated left lungs (mean, 6.5 ± 0.3 vs 7.5 ± 0.4; P = .04). ConclusionsIVLP successfully rehabilitated LPS-injured lungs compared to ECMO support alone in this preclinical porcine model.

Effect of HA330 resin-directed hemoadsorption on a porcine acute respiratory distress syndrome model

BackgroundBlood purification is an emerging approach to dampening the cytokine storm. This study aims to assess the efficacy of HA330 resin-directed hemoadsorption (HA) on endotoxin-induced porcine acute respiratory distress syndrome (ARDS) model.MethodsTwenty-four Chinese domestic pigs were allocated into saline group receiving intravenous infusion of saline (N = 6) and endotoxin group receiving intravenous infusion of LPS (N = 18). When ALI model was initially diagnosed, six pigs in the LPS and saline group were killed for BALF and histopathological analysis. The remaining 12 pigs in LPS group received 3-h HA (N = 6) or HA-sham (N = 6) treatment, respectively. Following another 5-h observation, animals were killed. Variables on hemodynamics, blood gases and lung mechanics were recorded at a series of time points. Differentially expressed cytokines and proteins were determined by ELISA and proteomics.ResultsHA treatment significantly improved injured oxygenation induced by LPS. HA also partially improved the barrier permeability and reduced lung edema and inflammation/injury induced by LPS infusion. Proteomic analysis showed the differentially expressed proteins between HA- and HA-sham-treated groups mostly belonged to the categories of acute inflammation/immune response, and proteolysis.ConclusionsHemoadsorption improved ARDS possibly by blunting the cytokine storm and by restoring homeostasis of the disordered proteome milieu in the exudative phase.

Bone Marrow-Derived Mesenchymal Stem Cells-Mediated Protection Against Organ Dysfunction in Disseminated Intravascular Coagulation Is Associated With Peripheral Immune Responses.

Disseminated intravascular coagulation (DIC) is a fatal thrombohemorrhagic disorder. Bone marrow-derived mesenchymal stem cells (BMSCs) are multipotent stem cells that have tremendous therapeutic effect. Our aim was to explore whether the immune mechanisms were associated with BMSCs-afforded protection against DIC. We generated a rat model of DIC by lipopolysaccharide (LPS, 3 mg/kg) injection via the tail vein. In the treatment group, rats were pre-treated with 1 × l03 , 1 × l04 , 1 × l05 , and 1 × l06 allogeneic BMSCs before LPS injection. Blood sample was withdrawn from the abdominal aorta at 0 (before), 4, and 8 h after LPS injection and used for biochemical analyses. After experiments, the mice were sacrificed and their organs were harvested and observed by H&E and PTAH staining. Continuous infusion of LPS into the rats gradually impaired the hemostatic parameters and damaged organ functions. However, pre-treatment with BMSCs dose-dependently improved the hemostatic parameters. Meanwhile, the treatment significantly suppressed the fibrin microthrombi formation and alleviated liver, heart, lung, and renal injuries. Flow cytometry analysis demonstrated that BMSCs pre-treatment inhibited LPS-induced upregulation of CD3+ CD8+ T cells and CD3+ /CD161a+ NKT cells in the peripheral blood. BMSCs pre-treatment reversed the upregualtion of the B-cell population and the percentage of CD43+ /CD172a+ monocytes in the DIC models. Finally, BMSCs pre-treatment decreased the levels of tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-1β (IL-1β), and interleukin-6 (IL-6) and increased the levels of interleukin-10 (IL-10) in LPS-induced DIC models. Pre-treatment with BMSCs can reduce coagulation and alleviate organ dysfunction via peripheral immune responses in LPS-induced DIC rat model. J. Cell. Biochem. 118: 3184-3192, 2017. © 2017 Wiley Periodicals, Inc.

Phytosterols Have Limited Direct Effect on Hepatocyte Transporter Expression but Synergize with Endotoxin to Enhance Kupffer Cell Inflammatory Response

Phytosterols, non‐nutritive components of soybean oil‐based lipid emulsions (SO), have been implicated in the development of parenteral nutrition associated liver disease (PNALD). Phytosterols may drive the pathogenesis of PNALD through a two‐hit mechanism: suppression of hepatic bile clearance through downregulation of the bile salt export pump (BSEP) and increased hepatic inflammation. Inflammation can further suppress BSEP and potentially the ATP‐binding cassette transporters ABCG5/8, the hepatic phytosterol clearance transporter, creating a feed‐forward loop leading to further hepatic injury. Two nuclear hormone receptors, Farnesoid X Receptor (FXR) and Liver X Receptor (LXR) regulate bile acid metabolism and have been shown to suppress the inflammatory response. The aim of our study was to first, determine if the hepatic transport dysfunction and inflammatory response is hepatocyte independent or requires activation of the resident macrophages, Kupffer cells; second, determine if ligand‐mediated activation of FXR and LXR can restore inflammation‐induced suppression of transporter function. Preterm piglets were administered parenteral nutrition for 10 d containing SO lipid emulsion. On day 10, piglets were given an 8‐h infusion of saline or LPS (10 μg*kg−1*h−1). In a separate experiment, primary hepatocytes and Kupffer cells were isolated from neonatal piglets. Cells were treated with media supplemented with 1% SO with or without the addition of a phytosterol mixture containing β‐sitosterol, stigmasterol, and campesterol. After incubation for 24 h, cells were additionally treated with FXR ligand Obeticholic acid (OCA), or LXR ligand GW3694 in the presence or absence of 50 ng/mL LPS or 10 ng/mL IL‐1β for 24 h. In piglets, administration of LPS lead to a significant suppression of BSEP and ABCG5/8 expression compared to saline infused piglets. In cell culture, administration of IL‐1β, but not LPS or phytosterols, led to suppression of both BSEP and ABCG5/8. In hepatocytes co‐treated with IL‐1β and OCA or GW3694, only BSEP expression was rescued. In Kupffer cells, phytosterols alone failed to activate expression of cytokines, however, when treated with both LPS and phytosterols increased cytokine expression significantly more than with LPS alone. From this study, we conclude inflammation can suppress the transporters for bile acid and phytosterol clearance. Our in vitro data shows that Kupffer cells are susceptible to enhanced activation of cytokine expression by phytosterols when cells are primed with LPS. These results suggest that phytosterols may contribute to the progression of liver disease through inflammation.Support or Funding InformationAmerican Society of Parenteral and Enteral Nutrition Rhoads Research Foundation, National Institute of Health Grant DK‐094616

Melatonin reduces changes to small intestinal microvasculature during systemic inflammation

BackgroundSystemic inflammation is known to impair the microcirculation in intestine and other organs as a result of multifactorial events. Here, we show that melatonin selectively reduces changes to the small intestinal microvasculature during systemic inflammation. Materials and methodsLipopolysaccharide (LPS) was infused at a rate of 0.5 mg/kg × h to induce systemic inflammation in male Wistar rats. Melatonin (single dose: 3 mg/kg × 15 min) was intravenously administered before as well as 120 and 240 min after the beginning of the LPS infusion. Systemic parameters were determined in regular intervals. Small intestine, liver, and kidney were histologically (structure of the microvessels, intravascular blood accumulation, and hemorrhages) and immunohistochemically (mast cells, granulocytes, and macrophages) analyzed. ResultsContinuous infusion of LPS resulted in dilated microvessels with intravascular blood accumulation (congestion) in liver and small intestine, the latter being particularly pronounced. Blood vessel walls remained intact, there were no hemorrhages. Melatonin significantly reduced these changes to the microvasculature in small intestine, but not in liver. It further reduced mast cell and granulocytes count in small intestine enhanced by LPS. However, except for the systemic blood pressure, melatonin neither improved LPS-dependent changes to systemic parameters nor mortality. ConclusionsChanges to the microvasculature during systemic inflammation are most pronounced in small intestine. Melatonin selectively diminishes these changes to small intestinal microvasculature, probably by reducing the local immune cells recruitment. However, changes to the small intestine are not decisive for the survival. We assume that the therapeutic benefit of melatonin is more likely in local intestinal inflammation.

Catalpol restores LPS-elicited rat microcirculation disorder by regulation of a network of signaling involving inhibition of TLR-4 and SRC.

LPS-induced microvascular hyperpermeability and hemorrhage play a key role in the development of sepsis, the attenuation of which might be an important strategy to prevent sepsis. However, the current clinical therapies have proven to be inefficient in improving the prognosis for patients with sepsis. Catalpol, an iridoid glycoside extracted from the roots of Rehmannia, has been reported to protect against LPS-induced acute lung injury through a Toll-like receptor-4 (TLR-4)-mediated NF-κB signaling pathway. However, it is still unknown whether catalpol can be an effective treatment to ameliorate the LPS-induced microvascular disorder. The present study aimed to investigate the impact of catalpol on LPS-induced mesenteric microvascular disorder and its underlying mechanism. Male Wistar rats were challenged by infusion of LPS (10 mg·kg-1·h-1) through the left femoral vein for 120 min. Post-treatment with catalpol (10 mg/kg) alleviated the LPS-induced microvascular hyperpermeability and hemorrhage; reduced mortality; ameliorated the alteration in the distribution of claudin-5 and the junctional adhesion molecule-1, as well as the degradation of collagen IV and laminin; and attenuated the increase of TLR-4 level, phosphorylations of Src tyrosine kinase, phosphatidyl inositol 3-kinase, focal adhesion kinase, and cathepsin B activation. In vitro study in human umbilical vein endothelial cells verified these results and further revealed that inhibition of TLR-4 and Src each simulated some, but not all, of the effects that catalpol exerted. Besides, surface plasmon resonance showed that catalpol could directly bind to TLR-4 and Src. These results demonstrated that catalpol was able to ameliorate the LPS-induced microvascular barrier damage and hemorrhage by targeting both TLR-4 and Src, thus attenuating the phosphorylation of Src kinase, phosphatidyl inositol 3-kinase, and focal adhesion kinase, as well as cathepsin B activation.

Exacerbates kidney injury in mice subjected to sepsis: critical impact of used heparin

Sepsis is the leading cause of death in critically ill patients, and the incidence of sepsis is increasing causes multiorgan failure, including acute kidney injury (AKI) and patients with both sepsis and AKI have an especially high mortality rate. Several different pathophysiological mechanisms have been proposed for sepsis-induced AKI: vasodilation-induced glomerular hypoperfusion, dysregulated circulation within the peritubular capillary network, inflammatory reactions by systemic cytokine storm or local cytokine production, and tubular dysfunction induced by oxidative stress animal sepsis models have been developed using LPS infusion. Renal dysfunction evaluated by serum creatinine and BUN was found in acute non-survivors (&lt;24 hours) and decreased urine output in subacute non-survivors (24–96 hours). The study show that increased AKI in animal with blood collected heparin (P=0.002) compared to citrate, furthermore; sham mice that received heparin did not develop AKI.

Repeated intrauterine infusions of lipopolysaccharide alter gene expression and lifespan of the bovine corpus luteum

Inflammation of the uterus is associated with disturbed ovarian function and reduced reproductive performance in dairy cows. To investigate the influence of endometritis on the bovine corpus luteum, 8 heifers received intrauterine infusions with either phosphate-buffered saline (PBS; 9mL) or Escherichia coli lipopolysaccharide (LPS; 3µg/kg of body weight diluted in 9mL of PBS) at 6-h intervals from 12h before and until 9d after ovulation during 2 cycles in a random order (ovulation=d 1). An untreated cycle was examined before and after PBS and LPS cycles, and the mean values from both untreated cycles were used as control. In all cycles, blood sampling and ultrasonography of the ovaries were performed on d 0, 1, 2, 4, 6, 8, 9, 10, 12, 15, 18, and then every 2d until ovulation. Endometrial cells were collected for cytology and quantitative real-time reverse transcriptase PCR on d 0, 6, and 9, and on d 0 and 6, respectively, and luteal tissue was collected for quantitative real-time reverse transcriptase PCR on d 6 and 9. Both, PBS and LPS infusions induced subclinical endometritis, which was accompanied by increased endometrial mRNA abundance of proinflammatory cytokines IL1β, IL8, and tumor necrosis factor α. Additionally, LPS challenge induced premature luteolysis, which was characterized by increased plasma concentrations of PGF2α metabolite, decreased plasma progesterone concentrations, and reduced luteal size and blood flow compared with the control. The luteal mRNA expression of the LPS receptor TLR4, PGE synthase, and the apoptosis-related factor CASP3 were higher, and those of steroidogenic factors STAR and HSD3B, the PGF receptor, and the angiogenic factor VEGFA121 were lower after LPS challenge compared with the control. In conclusion, repeated intrauterine LPS infusions during the first 9d of the estrous cycle alter gene expression and shorten the lifespan of the bovine corpus luteum.

Leucine ameliorates endotoxin‐induced alterations in protein‐protein interactions within mTORC1 complex in neonatal piglets

Sepsis decreases protein synthesis in skeletalmuscle in part by limiting the phosphorylation of translation initiationregulatory factors, eukaryotic initiation factor (eIF) 4E‐binding protein (4EBP1)and S6 kinase (S6K1), that are downstream of the mammalian target of rapamycincomplex 1 (mTORC1). Previous work inmature rats suggests that decreased raptor binding activity to eIF3b mayunderline the sepsis‐induced reduction in protein synthesis in skeletal muscle. Raptor‐eIF3b interaction is crucial for thestimulation of protein translation and its alteration during endotoxemia inmature animals appears to block the anabolic response to the branched‐chainamino acid, leucine (Leu). We have shown in healthy neonatal pigs that Leu supplementation increases muscle protein synthesis by enhancing the activation of mTORC1 and its downstream targets, 4EBP1 and S6K1. To determine if supplemental Leu can stimulate raptor‐eIF3b binding and muscle protein synthesis in theneonate during endotoxemia, overnight fasted neonatal pigs (n=6–8/group) were infused for 8 h with LPS or saline while plasma amino acids, glucose, and insulin were maintained at fasting levels during pancreatic‐substrate clamps. Leu (400 μmol·kg−1·hr−1) or saline was infused during the last h. Phosphorylation of 4EBP1 and S6K1, eIF4E‐eIF4G complex formation, raptor‐eIF3b binding, protein synthesis rates, ribosomal translational capacity (Cs) and translational efficiency (KRNA) were determined in longissimus dorsi (LD), gastrocnemius (Gast) and soleus muscles. LPS decreased protein synthesis and KRNA in LD, Gast, and soleus muscles (P&lt;0.05) while Cs did not change compared to controls. Leu infusion in the LPS group increased 4EBP1 and S6K1 phosphorylation, eIF4E‐eIF4G complex formation, KRNA, and the binding of raptor to eIF3b (by 2 to 3 fold) and protein synthesis (by over 80%) in LD, Gast, and soleus muscles compared to LPS infusion alone (P&lt;0.05). Compared to saline, Leu infusion increased protein synthesis, KRNA, raptor‐eIF3b interaction, 4EBP1 and S6K1 phosphorylation and eIF4E·eIF4G complex formation in all muscles (P&lt;0.05). Raptor‐eIF3b binding showed a linear correlation with 4EBP1 and S6K1 phosphorylation, eIF4E‐eIF4G formation, and protein synthesis in LD, Gast, and soleus muscles that was independent of treatment(P&lt;0.01). These findings suggest that parenteral Leu supplementation attenuates the reduction in muscle protein synthesis induced by endotoxemia by stimulating the interaction of raptor with eIF3 within the mTORC1 complex in the neonate.Support or Funding InformationSupported by NIH AR444474, USDA/ARS 6250‐51000‐055, NIH HD072891, and USDA NIFA 2013‐67015‐20438

Effects of Repeated LPS Infusion on Hematologic, Metabolic, and Intestinal Histology Parameters

Objectives were to characterize effects of repeated lipopolysaccharide infusion on circulating metabolites, white blood cells, and intestinal histology in pigs. Ten crossbred female pigs (168 ± 2 kg) were reproductively synchronized and jugular catheterized. The experimental period, during the follicular phase of the estrous cycle, lasted 5 d and pigs were assigned to either control (CON; 3 mL i.v. saline; n=4) or lipopolysaccharide (LPS; 0.1 μg/kg BW LPS i.v.; Escherichia coli 055:B5 Sigma, St. Louis, Missouri; n=6) with treatments administered 4x/d at 0000, 0600, 1200, and 1800h. Each animal was limit‐fed 2.7 kg of feed once daily at 0600 h. Blood samples for metabolites and a complete blood count were obtained daily at 1730h. Rectal temperatures were measured 2x/d at 0530 and 1730h and condensed into daily averages. Animals were sacrificed on d 6 beginning at 0600h and segments of jejunum, ileum, and colon were collected and analyzed for morphology and were stained for myeloperoxidase (a measure of neutrophil infiltration). Circulating platelets were decreased 31% (P&lt;0.01) and total white blood cell count tended to be decreased 10% (P=0.07) in LPS pigs relative to CON pigs. Lipopolysaccharide binding protein was increased 37% in LPS pigs relative to CON pigs (P=0.05) and was increased 60% on d 1 of the experiment relative to CON pigs (P = 0.02). There was a treatment by day interaction for neutrophils, lymphocytes, monocytes, and eosinophils where on d 1 only, neutrophils were increased 58% in LPS pigs relative to CON pigs (P&lt;0.01) and lymphocytes, monocytes, and eosinophils were decreased by 61, 93, and 81%, respectively in LPS pigs relative to CON pigs (P&lt;0.01 for all). Similar treatment by day differences in blood metabolites were observed where on d 1, insulin, blood urea nitrogen, β‐hydroxybutyric acid, and ionized calcium increased 36, 14, 13, and 7%, respectively, in LPS pigs relative to CON pigs (P=0.03, P=0.07, P=0.02, and P&lt;0.01, respectively). There was no treatment by day interaction for blood glucose; however, it tended to be increased 6% in LPS pigs on d 1 relative to CON pigs (84 vs 79 mg/dL; P=0.08). Rectal temperature did not differ between the two treatments throughout the entire study (38.5 vs 38.6°C; P=0.71). In the jejunum, a tendency for reduced goblet cell area was observed (2.2 vs 5.6%; P=0.06) as well as an increase in myeloperoxidase stained area (1.03 vs 0.87%; P&lt;0.01) in LPS pigs relative to CON pigs. No other differences in histological measurements were observed. In conclusion, LPS only altered metabolism on d 1 of the 5 d injection period. Platelets were the only measurement consistently affected throughout the 5 d. Jejunum was the only intestinal segment affected by repeated LPS exposure as determined by a tendency for reduced goblet cell area and an increase in myeloperoxidase stained area.

Intramammary lipopolysaccharide infusion alters gene expression but does not induce lysis of the bovine corpus luteum

Data from various studies indicate that the ovarian function in dairy cows can be compromised during intramammary infections. Therefore, in this study, we investigated if an experimentally induced mastitis has an effect on corpus luteum (CL) function in 14 lactating cows. On d 9 of the estrous cycle (d 1=ovulation), cows received a single dose of 200 μg of Escherichia coli lipopolysaccharide (LPS; dissolved in 10 mL of NaCL; n=8) or 10 mL of saline (control; n=6) into one quarter of the mammary gland. Measurements included plasma cortisol, haptoglobin, and progesterone (P4) concentrations, as well as luteal size (LTA) and relative luteal blood flow (rLBF). Sampling was performed on d 1, 4, and 8. On d 9, the main examination day, sampling was performed immediately before (0 h), every 1h (or at 3-h intervals for LTA and rLBF) until 9 h, as well as 12 and 24 h after treatment. Thereafter, measurements were taken on d 12, 15, 18, and then every 2 d until ovulation. Luteal tissue was collected for biopsy 24 h before and 6 h after treatment. Quantitative real-time PCR was applied to assess mRNA expression of steroidogenic factors (STAR, HSD3B), caspase 3, toll-like receptors (TLR2, -4), tumor necrosis factor α (TNFA), and prostaglandin-related factors (PGES, PGFS, PTGFR). Intramammary LPS infusion caused considerable inflammatory responses in the treated udder quarters. No decrease in plasma P4 concentrations was noted after LPS-challenge, and P4 levels did not differ between LPS-treated and control cows. Furthermore, LTA and rLBF values were not decreased after LPS challenge compared with the values obtained immediately before treatment. However, LPS infusion increased plasma levels of cortisol and haptoglobin compared with the control group. In the CL, mRNA abundance of TLR2 and TNFA was increased in cows after LPS-challenge (but not in control cows), whereas TLR4, steroidogenic, and prostaglandin-related factors remained similar to the mRNA abundance before treatment. In conclusion, intramammary LPS challenge induces systemic inflammatory reactions which alter the luteal mRNA abundance of TLR2 and TNFA but does not induce lysis of the CL.

Responses in whole-body amino acid kinetics to an acute, sub-clinical endotoxin challenge in lambs.

Some effects of parasitism, endotoxaemia or sepsis can be mitigated by provision of extra protein. Supplemented protein may encompass a metabolic requirement for specific amino acids (AA). The current study investigates a method to identify and quantify the amounts of AA required during inflammation induced by an endotoxin challenge. One of each pair of six twin sheep was infused in the jugular vein for 20 h with either saline (control) or lipopolysaccharide (LPS, 2 ng/kg body weight per min) from Escherichia coli. Between 12 and 20 h a mixture of stable isotope-labelled AA was infused to measure irreversible loss rates. From 16 to 20 h all sheep were supplemented with a mixture of unlabelled AA infused intravenously. Blood samples were taken before the start of infusions, and then continuously over intervals between 14 and 20 h. At 20 h the sheep were euthanised, and liver and kidney samples were taken for measurement of serine-threonine dehydratase (SDH) activity. LPS infusion decreased plasma concentrations of most AA (P<0·05; P<0·10 for leucine and tryptophan), except for phenylalanine (which increased P=0·022) and tyrosine. On the basis of the incremental response to the supplemental AA, arginine, aspartate, cysteine, glutamate, lysine (tendency only), glycine, methionine, proline, serine and threonine were important in the metabolic response to the endotoxaemia. The AA infusion between 16 and 20 h restored the plasma concentrations in the LPS-treated sheep for the majority of AA, except for glutamine, isoleucine, methionine, serine and valine. LPS treatment increased (P<0·02) SDH activity in both liver and kidney. The approach allows quantification of key AA required during challenge situations.

Acute Phase Proteins: How they portray Mastitis – A review

Acute Phase Proteins are blood proteins primarily synthesized by hepatocytes as part of the acute phase response (APR). APR to disease is accompanied by an increase in the circulating concentration of a number of plasma proteins which are collectively known as the Acute Phase Proteins. According to the concentration, APPs are classified in positive APP, if they increase or negative APP, if they decrease. During mastitis, APPs move from the systemic circulation to mammary gland or there could be de-novo synthesis of the APP within mammary gland tissues. Among the APPs, Serum Amyloid A (SAA), Haptoglobin (Hp), Alpha 1-acid glycoprotein and Lipopolysaccharide binding protein play a major role during mastitis in cattle and their concentration will increase with the severity of the infection, inflammation or trauma. SAA is involved in defense against Gram positive and Gram negative pathogens. SAA acts by modulating innate immune system and by acting as an opsonin. Among the SAA isoforms, mammary associated SAA3 (m-SAA3) is important. The blood concentration of SAA and Hp increases dramatically after LPS infusion, with SAA (acute and middle phase of APR) appearing before Hp (Late phase of APR). The herd level APP might be useful for determining the prevalence of clinical and subclinical infections indicated by the high serum concentration of selected APP and by serving as the prognostic tool. APPs, used as markers of animal health may possibly be influenced by environmental factors, handling procedures and other types of stress in the absence of disease.

Acute Phase Proteins: How they portray Mastitis – A review

Acute Phase Proteins are blood proteins primarily synthesized by hepatocytes as part of the acute phase response (APR). APR to disease is accompanied by an increase in the circulating concentration of a number of plasma proteins which are collectively known as the Acute Phase Proteins. According to the concentration, APPs are classified in positive APP, if they increase or negative APP, if they decrease. During mastitis, APPs move from the systemic circulation to mammary gland or there could be de-novo synthesis of the APP within mammary gland tissues. Among the APPs, Serum Amyloid A (SAA), Haptoglobin (Hp), Alpha 1-acid glycoprotein and Lipopolysaccharide binding protein play a major role during mastitis in cattle and their concentration will increase with the severity of the infection, inflammation or trauma. SAA is involved in defense against Gram positive and Gram negative pathogens. SAA acts by modulating innate immune system and by acting as an opsonin. Among the SAA isoforms, mammary associated SAA3 (m-SAA3) is important. The blood concentration of SAA and Hp increases dramatically after LPS infusion, with SAA (acute and middle phase of APR) appearing before Hp (Late phase of APR). The herd level APP might be useful for determining the prevalence of clinical and subclinical infections indicated by the high serum concentration of selected APP and by serving as the prognostic tool. APPs, used as markers of animal health may possibly be influenced by environmental factors, handling procedures and other types of stress in the absence of disease.

IgM-enriched solution BT086 improves host defense capacity and energy store preservation in a rabbit model of endotoxemia.

IntroductionThe therapeutic value of intravenous immunoglobulin (IVIG) as an adjuvant therapy in sepsis remains debatable. We hypothesized that intravenous administration of BT086, a predominantly IgM IVIG solution, would improve host defense in an established rabbit model of endotoxemia and systemic sepsis.MethodsNew Zealand white rabbits were randomized into the following four groups: (1) the negative control group without lipopolysaccharide (LPS, control), (2) the positive control group with LPS infusion (LPS group), (3) the albumin‐treated LPS group (ALB+LPS group), and (4) the BT086‐treated LPS group (BT086 + LPS group). A standardized amount of E. coli was intravenously injected into all of the animals. The vital parameters, the concentration of E. coli in the blood and other organs, the residual granulocyte phagocytosis activity, and the levels of the inflammatory mediators were measured. Histological changes in the lung and liver tissue were examined following autopsy.ResultsThe elimination of E. coli from the bloodstream was expedited in the BT086‐treated group compared with the LPS‐ and albumin‐treated groups. The BT086 + LPS group exhibited higher phagocytic activity of polymorphonuclear neutrophils (PMNs) than the control and ALB+LPS groups. The liver energy stores were higher in the BT086 + LPS group than in the other groups.ConclusionOur data suggest that the IgM‐enriched IVIG has the potential to improve host defense in a rabbit model of endotoxemia. Studies using different animal models and dosages are necessary to further explore the potential benefits of IgM‐enriched IVIG solutions.

Inhibitory effect of suramin on inflammatory response in pulmonary tissue and peripheral blood in LPS-induced septic mice

To investigate the effect of suramin on inflammatory response in pulmonary tissue and peripheral blood in septic mice. Twenty-four male C57BL/6 mice were randomly divided into two groups, and suramin(5 mg/kg) or normal saline was intravenously injected 30 min before LPS(5 mg/kg)infusion, respectively. The contents of TNF-α and IL-6 in pulmonary tissue and peripheral blood were detected by ELISA. Suramin or saline-pretreated human mononuclear THP-1 cells were treated with 100 ng/mL LPS in vitro. The expression of TNF-α and IL-6 mRNA and the activity of NF-κB were analyzed by quantitative PCR and Western blotting at different time points after LPS treatment, respectively. Compared with the saline group, the TNF-α and IL-6 levels in pulmonary tissue and peripheral blood were significantly reduced in suramin group at 24 h after LPS treatment(all P<0.01); while there was no significant difference at 72 h between two groups(all P>0.05). The expression of TNF-α, IL-6 mRNA and the activity of NF-κB was decreased in suramin group at different time points after LPS treatment. Suramin can protect LPS-induced acute lung injury through down-regulation of systemic and pulmonary pro-inflammatory factors, which may be associated with the inhibition of NF-κB activity.

Peripheral inflammation increases seizure susceptibility via the induction of neuroinflammation and oxidative stress in the hippocampus.

BackgroundNeuroinflammation with activation of microglia and production of proinflammatory cytokines in the brain plays an active role in epileptic disorders. Brain oxidative stress has also been implicated in the pathogenesis of epilepsy. Damage in the hippocampus is associated with temporal lobe epilepsy, a common form of epilepsy in human. Peripheral inflammation may exacerbate neuroinflammation and brain oxidative stress. This study examined the impact of peripheral inflammation on seizure susceptibility and the involvement of neuroinflammation and oxidative stress in the hippocampus.ResultsIn male, adult Sprague-Dawley rats, peripheral inflammation was induced by the infusion of Escherichia coli lipopolysaccharide (LPS, 2.5 mg/kg/day) into the peritoneal cavity for 7 days via an osmotic minipump. Pharmacological agents were delivered via intracerebroventricular (i.c.v.) infusion with an osmotic minipump. The level of cytokine in plasma or hippocampus was analyzed by ELISA. Redox-related protein expression in hippocampus was evaluated by Western blot. Seizure susceptibility was tested by intraperitoneal (i.p.) injection of kainic acid (KA, 10 mg/kg). We found that i.p. infusion of LPS for 7 days induced peripheral inflammation characterized by the increases in plasma levels of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). This is associated with a significant increase in number of the activated microglia (Iba-1+ cells), enhanced production of proinflammatory cytokines (including IL-1β, IL-6 and TNF-α), and tissue oxidative stress (upregulations of the NADPH oxidase subunits) in the hippocampus. These cellular and molecular responses to peripheral inflammation were notably blunted by i.c.v. infusion of a cycloxygenase-2 inhibitor, NS398 (5 μg/μl/h). The i.c.v. infusion of tempol (2.5 μg/μl/h), a reactive oxygen species scavenger, protected the hippocampus from oxidative damage with no apparent effect on microglia activation or cytokine production after peripheral inflammation. In the KA-induced seizure model, i.c.v. infusion of both NS398 and tempol ameliorated the increase in seizure susceptibility in animals succumbed to the LPS-induced peripheral inflammation.ConclusionsTogether these results indicated that LPS-induced peripheral inflammation evoked neuroinflammation and the subsequent oxidative stress in the hippocampus, resulting in the increase in KA-induced seizure susceptibility. Moreover, protection from neuroinflammation and oxidative stress in the hippocampus exerted beneficial effect on seizure susceptibility following peripheral inflammation.