Spontaneous Oxidative Burst Research Articles

Structural Features and Immunomodulatory Effects of Water-Extractable Polysaccharides from Macrolepiota procera (Scop.) Singer.

Macrolepiota procera (MP) is an edible mushroom used in the treatment of diabetes, hypertension and inflammation. However, the structure and biological effects of its polysaccharides (PSs) are unclear. This study investigates the structural features of a PS complex from MP (MP-PSC), its immunomodulatory activities and effects on probiotic and pathogenic bacteria. MP-PSC was obtained by boiling water, and PSs were characterized by 2D NMR spectroscopy. The immunomodulatory effects on blood and derived neutrophils, other leukocytes, and murine macrophages were studied by flow cytometry, chemiluminescence, spectrophotometry, and ELISA. The total carbohydrate content of MP-PSC was 74.2%, with glycogen occupying 36.7%, followed by β-D-glucan, α-L-fuco-2-(1,6)-D-galactan, and β-D-glucomannan. MP-PSC (200 μg/mL) increased the number of CD14+ monocyte cells in the blood, after ex vivo incubation for 24 h. It dose-dependently (50–200 μg/mL) activated the spontaneous oxidative burst of whole blood phagocytes, NO, and interleukin 6 productions in RAW264.7 cells. MP-PSC exhibited a low antioxidant activity and failed to suppress the oxidative burst and NO generation, induced by inflammatory agents. It (2.0%, w/v) stimulated probiotic co-cultures and hindered the growth and biofilm development of Escherichia coli, Streptococcus mutans and Salmonella enterica. MP PSs can be included in synbiotics to test their immunostimulating effects on compromised immune systems and gut health.

Neutrophil Respiratory Burst: an immunological indicator of post stroke infection

Stroke is long known to be followed by a series of immunosuppressive events, and infections might be a cause of death after an acute insult of stroke. The aim of our work was to assess the percentage of neutrophils showing spontaneous oxidative burst in patients with acute ischemic stroke. The study included 30 patients with acute cerebral infarction subjected to the following: magnetic resonance imaging of the brain immediately on admission, and blood sampling on day one of admission (baseline) and after 3 days of admission. Blood samples were used for the assessment of: differential leucocyte count and percentage of neutrophils showing spontaneous oxidative burst, performed by flow cytometry. Thirty age and gender matched controls were also recruited. Neutrophil respiratory burst percentage was significantly lower in stroke patients in comparison to controls (P < 0.001), and stroke patients had significantly lower neutrophil respiratory burst percent on day 3 of admission compared to the baseline (P < 0.001). Stroke-induced immune alterations including impairment of the first-line defense performed by neutrophils against bacteria. The hypothesis that these changes enhance susceptibility to acquired infections is supported by our observation that oxidative burst in neutrophils was more impaired in patients with stroke who exhibited subsequent stroke-associated infections.

Activation and Functional Priming of Blood Neutrophils in Non-Alcoholic Fatty Liver Disease Increases in Non-Alcoholic Steatohepatitis.

IntroductionIn non-alcoholic fatty liver disease (NAFLD), neutrophils in liver infiltrates are activated, which may contribute to disease progression towards non-alcoholic steatohepatitis (NASH). However, the functional status of the blood neutrophils remains unknown and their role in the disease mechanisms is thus uncertain. We therefore characterized activation and function of blood neutrophils in patients with NAFLD in relation to clinical disease markers and the NAFLD plasma milieu.MethodsWe studied 20 patients with NAFLD, among these 6 patients with NASH, and 14 healthy persons. Neutrophil activation, interleukin (IL)-8 production and oxidative burst were measured by flow cytometry on participants´ neutrophils and on healthy neutrophils exposed in vitro to plasma from the study participants.ResultsBlood neutrophils from the NASH patients showed a doubling in their expression of the activation marker CD62L. Also, all NAFLD patients had 50–100% increased expression of CD11b. Functionally, NASH neutrophils had 30% elevated IL-8 production and more than doubled spontaneous oxidative burst. In all NAFLD patients, higher spontaneous oxidative burst was associated with worse liver function. Incubation of healthy neutrophils with NAFLD plasma paradoxically slightly reduced CD62L and CD11b expression, and NASH plasma also reduced the frequency of IL-8-producing neutrophils.ConclusionIn NAFLD, blood neutrophils are activated, and in NASH also functionally primed. This suggests a progressive neutrophil aggressiveness already present with liver fat infiltration. However, NAFLD plasma in vitro, if anything, had the opposite effect on the healthy neutrophils so the NAFLD-related neutrophil activation cannot be attributed to humoral factors and remains unexplained.

The effect of ammonia on canine polymorphonuclear cells.

Hyperammonaemia is a common complication of liver disease in dogs. High concentrations of ammonia can be detrimental to dogs with liver disease for several reasons, notably by causing hepatic encephalopathy (HE) which describes the wide range of neurological abnormalities ranging from altered behaviour to seizures that are well recognised complications in dogs with hepatic disorders. In human patients with liver disease, hyperammonaemia has also been linked to the development of other systemic complications such as dysregulation of the innate immune system. In contrast, the effects of hyperammonaemia on the canine innate immune system is currently unknown. The aim of this study was to investigate the effects of ammonia on the oxidative burst activity of canine polymorphonuclear cells in vitro. Blood obtained from healthy dogs (n = 8) was incubated with escalating concentrations of ammonia ranging from 0 to 250μM, and the percentage of cells experiencing an oxidative burst was evaluated using a commercial kit (Phagoburst™) and flow cytometry. The spontaneous oxidative burst was evaluated without stimulation and also following stimulation with E coli. The pH of the blood was also measured at the differing ammonia concentrations. There was an increase in the percentage of cells experiencing a spontaneous oxidative burst from ammonia concentrations of 125μM (p = <0.05) and above (p = <0.01), with a 4.9 fold increase at 200μM (p = < 0.001). In those cells stimulated with E coli, incubation with increasing ammonia concentrations did not result in a significant difference in oxidative burst from baseline (p = 0.953). There was no statistically significant difference between the pH of the blood at the various ammonia concentrations (p = 0.2) suggesting that the difference in spontaneous oxidative burst was due to the ammonia rather than simply a change in pH conditions. In summary, the spontaneous oxidative burst of neutrophils was significantly increased from baseline. This supports a potential role of ammonia in contributing to innate immune system dysfunction in dogs with liver disease, and may present a future therapeutic target.

Neutrophil Toll-Like Receptor 9 Expression and the Systemic Inflammatory Response in Acetaminophen-Induced Acute Liver Failure.

There is a marked propensity for patients with acetaminophen-induced acute liver failure to develop sepsis, which may culminate in multiple organ failure and death. Toll-like receptors sense pathogens and induce inflammatory responses, but whether this is protective or detrimental in acetaminophen-induced acute liver failure remains unknown. We assessed Toll-like receptor expression on circulating neutrophils and their function in 24 patients with acetaminophen-induced acute liver failure and compared with 10 healthy controls. Neutrophil Toll-like receptor 2, -4, and -9 expression and cytokine production and function were studied ex vivo at baseline and following stimulation with lipopolysaccharide, oligodeoxynucleotides, ammonium chloride, and interleukin-8. To examine the influence of acetaminophen-induced acute liver failure plasma and endogenous DNA on Toll-like receptors-9 expression, healthy neutrophils were incubated with acetaminophen-induced acute liver failure plasma with and without deoxyribonuclease-I. Circulating neutrophil Toll-like receptor 9 expression was increased in acetaminophen-induced acute liver failure on day 1 compared with healthy controls (p = 0.0002), whereas Toll-like receptor 4 expression was decreased compared with healthy controls (p < 0.0001). Toll-like receptor 2 expression was unchanged. Neutrophil phagocytic activity was decreased, and spontaneous oxidative burst increased in all patients with acetaminophen-induced acute liver failure compared with healthy controls (p < 0.0001). Neutrophil Toll-like receptor 9 expression correlated with plasma interleukin-8 and peak ammonia concentration (r = 0.6; p < 0.05) and increased with severity of hepatic encephalopathy (grade 0-2 vs 3/4) and systemic inflammatory response syndrome score (0-1 vs 2-4) (p < 0.05). Those patients with advanced hepatic encephalopathy (grade 3/4) or high systemic inflammatory response syndrome score (2-4) on day 1 had higher neutrophil Toll-like receptor 9 expression, arterial ammonia concentration, and plasma interleukin-8 associated with neutrophil exhaustion. Healthy neutrophil Toll-like receptor 9 expression increased upon stimulation with acetaminophen-induced acute liver failure plasma, which was abrogated by preincubation with deoxyribonuclease-I. Intracellular Toll-like receptor 9 was induced by costimulation with interleukin-8 and ammonia. These data point to neutrophil Toll-like receptor 9 expression in acetaminophen-induced acute liver failure being mediated both by circulating endogenous DNA as well as ammonia and interleukin-8 in a synergistic manner inducing systemic inflammation, neutrophil exhaustion, and exacerbating hepatic encephalopathy.

Liposomes as carriers of the lipid soluble antioxidant resveratrol: Evaluation of amelioration of oxidative stress by additional antioxidant vitamin

AimResveratrol (RES) is a well-known antioxidant, yet in combination with other antioxidant vitamins, it was found to be more effective than any of these antioxidants alone. Present work aims to compare the antioxidant actions of resveratrol with and without vitamin C following delivery as liposomes tested using chemical and cellular antioxidative test systems. Main methodsLiposomes were prepared by the thin film hydration method and characterised for percent drug entrapment (PDE), Z-average mean size (nm), polydispersity index (PDI) and zeta potential. Antioxidative capacity was determined by studying the inhibition of AAPH induced luminol enhanced chemiluminescence and inhibition of ROS production in isolated blood leukocytes. Intracellular oxygen-derived radicals were measured using flow cytometry with buffy coats (BC) and human umbilical vein endothelial cells using H2DCF-DA dye. Key findingsParticle size varied from 134.2±0.265nm to 103.3±1.687nm; PDI ≤0.3; zeta potential values were greater than −30mV and PDE ≥80%. Radical scavenging effect was enhanced with liposomal systems; oxidative burst reaction in BC was inhibited by liposomal formulations, with the effect slightly enhanced in presence of vitamin C. Reduction in reactive oxygen species (ROS) production during spontaneous oxidative burst of BC and incubation of HUVECs with H2O2 further intensified the antioxidative effects of pure RES and liposomal formulations. SignificanceThe present work clearly shows that the antioxidative effects of resveratrol loaded into liposomes are more pronounced when compared to pure resveratrol. Liposomal resveratrol is even active within the intracellular compartments as RES could effectively quench the intracellular accumulation of ROS.

PTU-040 Alcohol: always detrimental to the immune system? The role of active alcohol consumption on neutrophil function in alcohol-related cirrhosis

IntroductionNeutrophil dysfunction has been reported in patients with alcohol-related cirrhosis (ARC) and is associated with increased risk of infection and mortality. There is a paucity of understanding regarding the mechanisms...

P52 Neutrophil dysfunction: a potential biomarker of poor prognosis in acute liver failure?

IntroductionIn acute liver failure (ALF) an exaggerated systemic inflammatory response can result in neutrophil activation with subsequent tissue damage from release of proteolytic enzymes and reactive oxygen species, contributing to...

PWE-056 A compensatory anti-inflammatory response syndrome triggered by neutrophil-induced oxidative stress is associated with chronic low grade hepatic encephalopathy in patients with advanced cirrhosis

Introduction The systemic inflammatory response syndrome (SIRS) exacerbates hepatic encephalopathy (HE) in patients with cirrhosis. SIRS is a proinflammatory response tasked with killing infectious organisms through activation of the immune system, whereas CARS is a global deactivation of the immune system aimed at restoring homeostasis. The timing and magnitude of CARS may impact on patient outcomes. This study aimed to characterise the relationship between neutrophil oxidative stress, plasma cytokine profile and ammonia in a cohort of patients with advanced cirrhosis undergoing assessment for liver transplantation (LT). Patients specifically referred for LT with chronic HE (n=7) were compared to those who had no history of overt HE (n=17) and healthy controls (HC) (n=8). Methods Neutrophils were isolated at the time of LT assessment and 72 h following LT (n=6/24). Quantification of spontaneous oxidative burst (OB) was determined by measuring the percentage of neutrophils producing reactive oxidants at rest. Formation of oxidative reactants was monitored by oxidation of dihydrorhodamine (DHR 123) to rhodamine. To identify neutrophils, cells were stained with anti- CD16-PE IgG1 and analysed by flow cytometry. Clinical data, blood biochemistry, arterial ammonia and a full set of microbial cultures were collected. Cytokine analysis for TNF-α, IL-1s, IL-6, IL-8, IL-10 and IL-17 were performed by ELISA. Results There were no differences in MELD score, neutrophil count, ammonia (p=0.85) or empirical antibiotic use between those with chronic HE and those without. Neutrophil spontaneous OB was elevated in those with HE (30% (9–77) vs 13% (9–41), p=0.04) compared to those without. Patients with chronic HE had markedly decreased proinflammatory cytokines (TNF-α, p=0.02; IL-6, p=0.009 and IL-17, p=0.01) compared to those without HE but not the neutrophil-specific cytokine IL-8, upregulated in response to oxidative stress. The anti-inflammatory cytokine IL-10 was low in patients with HE compared to those without HE (6 (5–65) vs 207 (34–369) pg/ml, p=0.02) and HCs. Cirrhotic cytokine profiles and elevated spontaneous OB returned to HC levels post-LT. Conclusion These data suggest that patients with advanced cirrhosis and chronic HE have neutrophil-induced oxidative stress which triggers CARS rather than SIRS with time. IL-10 may protect against chronic HE and oxidative stress.

Ammonia impairs neutrophil phagocytic function in liver disease†

Hyperammonemia is a feature of liver failure, which is associated with increased risk of infection. The aims of the present study were to determine in vitro, in rats fed an ammoniagenic diet and in patients with cirrhosis, whether induction of hyperammonemia results in neutrophil dysfunction. As hyperammonemia produces cell swelling, we explored the role of the osmoregulating, p38 mitogen-activated protein kinase (p38(MAPK)) pathway in mediating this neutrophil dysfunction. Neutrophils were isolated from blood of healthy volunteers and incubated with either 75 microM ammonia or phosphate-buffered saline. Both groups were studied under hyponatremic conditions and/or with the addition of p38(MAPK) modulators. Neutrophil phagocytosis was measured in naive rats and rats fed an ammoniagenic diet and in patients with stable cirrhosis given placebo (n = 8) or an amino acid solution inducing hyperammonemia (n = 8). Cell volume and phagocytosis was analyzed by fluorescent-activated cell sorting using fluorescein isothiocyanate-labeled E. coli. p38(MAPK) phosphorylation was measured by western blotting. In healthy neutrophils incubated with ammonia and in rats fed an ammoniagenic diet, neutrophils showed evidence of swelling, impaired phagocytosis, and increased spontaneous oxidative burst compared to controls. Phagocytosis was significantly impaired in patients with induced hyperammonemia compared to placebo. The effects of hyperammonemia and hyponatremia were synergistic. The p38(MAPK) intracellular signaling pathways were activated in healthy neutrophils exposed to ammonia in association with increased burst activity. Neutrophil phagocytic dysfunction was abrogated by the addition of a p38(MAPK) agonist. Ammonia produces neutrophil swelling and impairs neutrophil phagocytosis. The p38(MAPK) intracellular signaling pathway has been shown to be important in mediating the ammonia-induced neutrophil dysfunction.

CD4-EXPRESSING CELLS ARE EARLY MEDIATORS OF THE INNATE IMMUNE SYSTEM DURING SEPSIS

It is well established that the immune response to sepsis is mediated by leukocytes associated with the innate immune system. However, there is an emerging view that T lymphocytes can also mediate this response. Here, we observed a significant depletion of both CD4 and CD8 T cells in human patients after blunt trauma. To determine what effect the loss of these cells may have during a subsequent infection, we obtained CD4- and CD8-deficient mice and subjected them to cecal ligation and puncture (CLP). We observed that CD4 knockout (KO) mice showed increased CLP-induced mortality compared with CD8-deficient and wild-type (WT) mice especially within the first 30 h of injury. CD4 KO mice also exhibited significantly increased IL-6 concentrations after the CLP. The CD4 KO mice had an increased concentration of bacteremia as compared with WT mice. Antibiotic treatment decreased mortality in the CD4 KO mice as compared with no changes in the wild mice after CLP. Neutrophils isolated from septic CD4 KO mice showed decreased spontaneous oxidative burst compared with neutrophils taken from septic controls. We examined the role of IFN-gamma by using mice deficient in this cytokine and found these mice to have significantly higher mortality as compared with WT mice. Finally, we detected a 2-fold increase in CD11b+ cells that exhibited intracellular IFN-gamma staining in the peritoneum of WT mice after CLP. The data suggest that CD4+ cells may facilitate the early clearance of bacteria by regulating neutrophils function possibly through an IFN-gamma-dependent mechanism.

Ron receptor tyrosine kinase-dependent hepatic neutrophil recruitment and survival benefit in a murine model of bacterial peritonitis

To determine whether Ron receptor tyrosine kinase signaling affects the in vivo response to bacterial peritonitis. Experimental study. University laboratory. Male mice 8-11 wks of age (22-28 g). A genetic approach comparing wild-type mice to mice with a targeted deletion of the Ron tyrosine kinase signaling domain (TK-/-) was undertaken to determine the influence of Ron receptor in the in vivo response to a well-characterized model of bacterial peritonitis and sepsis induced by cecal ligation and puncture. Several clinical (i.e., survival curves, blood and tissue bacterial burdens, and neutrophil oxidative burst), morphologic (i.e., liver histology and leukocyte trafficking), and biochemical variables (i.e., serum aminotransferases and select serum cytokine and chemokine levels) important for assessing inflammatory responses to bacterial infection were assessed in mice following cecal ligation and puncture. Ron TK-/- mice had a significant decrease in survival time compared with controls, and this was associated with a significant increase in bacterial colony-forming units found in the blood and several end-organs. Moreover, this increased bacterial load was associated with increased liver necrosis and serum alanine aminotransferase levels. Neutrophils isolated from TK-/- mice exhibited decreased spontaneous oxidative burst capacity ex vivo, and by intravital microscopy, a reduced level of neutrophil migration to and translocation within the liver was observed. Loss of Ron signaling resulted in significantly reduced production of serum monocyte chemoattractant protein-1 and interleukin-6 levels following cecal ligation and puncture, and peritoneal macrophage isolated from TK-/- mice exhibited blunted production of monocyte chemoattractant protein-1, interleukin-6, and macrophage inflammatory protein-2 following stimulation with endotoxin ex vivo. Ron signaling negatively regulates the response to polymicrobial infection by regulating the activation and recruitment of inflammatory cells necessary for clearing a systemic bacterial burden. This effect may be regulated in part through the Ron-dependent, macrophage-mediated production of cytokines and chemokines, namely monocyte chemoattractant protein-1, interleukin-6, and macrophage inflammatory protein-2, important for neutrophil mobilization.

RON RECEPTOR SIGNALING ENHANCES SURVIVAL DURING INFECTIONS BY STIMULATING MACROPHAGE-DEPENDENT PRO-INFLAMMATORY MEDIATOR PRODUCTION

The Ron receptor tyrosine kinase is expressed rather ubiquitously in most cell types including epithelial cells and peritoneal macrophage. Very little is known regarding the biology of the membrane bound Ron receptor tyrosine kinase in acute inflammation and during bacterial infections, either in vivo or in vitro. Objective: To elucidate the role of Ron receptor tyrosine kinase during non-sterile sepsis. Methods: Mice were subjected to a CLP using a 22-gauge needle (single punch) followed by a 95% ligation of the cecum. Results: Our data indicate that Ron receptor activity enhances survival following CLPinduced injury. In this model, Ron signaling augments bacterial clearance and protects against liver damage. Moreover, Ron signaling increases septic neutrophil spontaneous oxidative burst. These Ron-dependent activities are associated with elevations of IL-6, MCP-1, and MIP-2, through a mechanism likely associated with STAT-3 phosphorylation. Conclusion: The actions of the Ron receptor tyrosine kinase are beneficial during sepsis. The data suggest that Ron activity on the macrophage enhances generation of neutrophil activating mediators. This, in turn, limits bacterial spread and this likely results in the observed increased survival and mean survival times. Support: SHC Project 8560 (CC), NIH GM072760 (CC), SHC Project 8950 (SW), and NIH DK0735552 (SW).

Lymphocyte Subpopulations, Oxidative Burst and Apoptosis in Peripheral Blood Cells of Patients with Multiple Sclerosis–Effect of Interferon-β

At present, the most efficient therapeutical treatment of multiple sclerosis (MS) is achieved by IFN-β. However, its in vivo effects remain incompletely understood. If applied parenterally, the hydrophobic IFN-β acts primarily on blood cells with probable selectivity for functionally different lymphocyte subpopulations, monocytes and granulocytes. We have investigated the expression of the activation marker interleukin-2 receptor-α (CD25) on CD3+ T cells, CD19+ B cells, foetal-type γδ+CD3+ T cells and foetal-type CD5+CD19+ B cells of the peripheral blood. In addition, the oxidative burst activity and apoptosis have been determined in mononuclear and polymorphonuclear blood cells, respectively. The study accompanied a phase III trial with IFN-β1b (BETAFERON®, Schering). Two groups of MS patients with relapsing-remitting course of the disease have been investigated at 8 time points (days 0, 5, 15, 31, 60, 90, 180 and 270 after starting therapy): (1) verum group (n=8) with application of 8 Mill. units IFN-β1b every other day, and (2) placebo group (n=4) with application of placebo for 3 months and therapy as in (1) from day 90 onward. The main results were: (1) Activated T cells decreased until day 180 in the verum group and return thereafter to pre-treatment values, whereas in the placebo group the values remained relatively stable over the whole observation period. (2) Activated B cells increased between days 90 and 270 in both groups, i.e. after verum application in both groups. (3) Foetal-type B cells were more activated than total B and T cells with increase over time in both groups. (4) Foetal-type T cells exerted relatively stable intra-individual levels with generally low CD25 expression, but punctual CD25 peaks in both groups. (5) The spontaneous oxidative burst was higher in lymphocytes, more variable in monocytes and faster increasing in granulocytes in the verum group than in the placebo group. (6) Apoptosis of mononuclear cells and granulocytes showed similar variations in the verum and placebo groups with the exception of a selective increase over time of the proportion of granulocytes undergoing induced apoptosis in the verum group. It is concluded that IFN-β has the following main effects on the immune system of MS patients: (1) the T cell immunity is systemically and reversibly suppressed, (2) the foetal-type lymphocytes, which are responsible for the first line of defence of infections, are stimulated in the long range, (3) the oxidative burst activity is increased in lymphocytes and granulocytes and instable in monocytes, and (4) the inducibility of apoptosis in granulocytes is increased. Re-examination of the altered blood cell parameters after long-term IFN-β therapy is warranted.

Pathogenicity of selected Toxoplasma gondii isolates in young pigs

The pathogenicity in 7-week-old pigs to five different Toxoplasma gondii strains of various host species origin was compared after i.v. inoculation of 10 4 tachyzoites. Additionally, one group of pigs was inoculated i.v. with 10 6 tachyzoites of the reference strain, SSI 119. In response to the infection a significant effect of T. gondii tachyzoite inoculation dose as well as differences among strains could be observed in several parameters. The 10 6-dose inoculated pigs showed variable degrees of clinical illness and recurrent episodes of fever 4–17 days p.i., while pigs of four of the 10 4 tachyzoite inoculated groups experienced a short-lived rise in body temperature from day 6–8 p.i. without any apparent illness or inappetence. Control pigs and pigs infected with the least pathogenic strain had normal body temperature throughout the experiment. In all inoculated pigs, T. gondii-specific IgM and IgG antibodies appeared from day 8–10 and 10–17 p.i., respectively. Serum levels of alkaline phosphatase and the acute phase protein haptoglobin were decreased or increased, respectively, in response to the infection. Differential leukocyte count on peripheral blood revealed a significant lymphocytopenia on day 6 p.i. equal to both CD4 + and CD8 + T-cells, but shifting towards a reduced ratio of CD4 +/CD8 + T-cells from day 8–14 p.i. In the 10 6-dose inoculated pigs a considerable increase in zymosan induced and spontaneous oxidative burst capacity of peripheral blood leukocytes was observed from 6 days p.i. compared with control pigs. Oxidative burst capacity was not examined for other pigs. In conclusion, several useful parameters to identify differences in T. gondii pathogenicity other than mortality were identified. Furthermore, even at low doses, significant differences between recently collected Danish T. gondii field isolates were demonstrated after i.v. inoculation in young pigs.