Reactive Oxygen Species Production In Monocytes Research Articles

α-Iso-cubebene attenuates neointima formation by inhibiting HMGB1-induced monocyte to macrophage differentiation via suppressing ROS production

α-Iso-cubebene (ICB) is a dibenzocyclooctadiene lignin contained in Schisandra chinensis, a medicinal herb used to improve cardiovascular symptoms. To investigate the mechanisms involved, the effects of ICB on cellular production of reactive oxygen species (ROS) was determined using cultured human THP-1 cells. When THP-1 cells were stimulated with HMGB1, cellular concentration of ROS was increased in dose- and time-dependent manners. These increases were significantly attenuated in cells pretreated with NADPH oxidase inhibitors, diphenyleneiodonium chloride and apocynin, but not by other inhibitors related to ROS generation in monocytes. The expression of constitutively expressed NADPH oxidase (NOX) subunits including NOX1, NOX2, NOX4 and NOX5 was not affected by HMGB1, but HMGB1-induced ROS production was exclusively attenuated in NOX2-deficient cells using siRNA, suggesting an enhanced NOX2 complex assembly. When cells were stimulated with HMGB1, p47phox phosphorylation at ser345, ser359 and ser370 was increased in dose- and time-dependent manners, which were significantly attenuated in ICB (3–10 μg/mL)-pretreated cells. In addition, HMGB1-induced monocyte–macrophage differentiation (MMD) in bone marrow-derived cells isolated from mice were significantly attenuated in cells treated with apocynin and ICB. Also, macrophage infiltration and intimal hyperplasia in the wire-injured femoral artery were significantly attenuated in ICB-treated mice compared to wild-type control mice. The results of this study show that ICB inhibits HMGB1-induced MMD by suppressing ROS production in monocytes, thus suggest that ICB has therapeutic potential for vascular inflammation with subsequent intimal hyperplasia related to vascular injury.

Bacterial species-specific modulatory effects on phenotype and function of camel blood leukocytes

BackgroundRecent studies have reported pathogen-species-specific modulating effects on the innate immune system. Escherichia coli, Staphylococcus aureus, and Streptococcus agalactiae are important pathogenic bacteria responsible for different infectious diseases in several animal species. In the present study, a whole blood culture with S. aureus, E. coli, or S. agalactiae and flow cytometry were used to investigate, whether stimulation with different bacterial species induces different immunomodulation patterns in camel leukocytes. The expression of different cell surface myeloid markers and cell adhesion molecules on monocytes and neutrophils was investigated. In addition, the capacity of monocytes and neutrophils to produce reactive oxygen species (ROS) was analyzed.ResultsStimulation with either of the bacterial species resulted in the expansion of the camel CD14highMHCIIhigh monocyte subset with a reduced fraction of CD14highMHCIIlow monocytes. For the CD14lowMHCIIhigh monocytes, however, only stimulation with S. aureus or S. agalactiae increased their fractions in blood. Although all bacterial species elicited the upregulation of cell surface MHC class II molecules on granulocytes, the increase was, however, highest on cells stimulated with S. aureus. The expression levels of the two adhesion molecules, CD11a and CD18, on neutrophils and monocytes were differently affected by bacterial stimulation. Functionally, E. coli failed to stimulate ROS production in monocytes, while induced a strong ROS production response in granulocytes. S. agalactiae elicited a week ROS production in granulocytes when compared to the other two pathogens.ConclusionsThe different responsiveness of monocytes and granulocytes toward different bacterial species indicates different host-pathogen interaction mechanisms for the two cell populations. In addition, the phenotypic and functional differences between cells stimulated with E. coli, S. aureus, or S. agalactiae suggests pathogen-species-specific modulating effects of the bacterial pathogens on the camel innate myeloid cells.

Alpha-cyclodextrin inhibits cholesterol crystal-induced complement-mediated inflammation: A potential new compound for treatment of atherosclerosis

Background and aimsCholesterol crystal (CC)-induced inflammation is a critical step in the development of atherosclerosis. CCs activate the complement system and induce an inflammatory response resulting in phagocytosis of the CCs, production of reactive oxygen species (ROS) and release of cytokines. The cyclodextrin 2-hydroxypropyl-β-cyclodextrin has been found to reduce CC-induced complement activation and induce regression of established atherosclerotic plaques in a mouse model of atherosclerosis, thus inhibition of complement with cyclodextrins is a potential new strategy for treatment of inflammation during atherosclerosis. We hypothesized that other cyclodextrins, like α-cyclodextrin, may have related functions. MethodsThe effect of cyclodextrins on CC-induced complement activation, phagocytosis, and production of ROS from granulocytes and monocytes was investigated by flow cytometry and ELISA. ResultsWe showed that α-cyclodextrin strongly inhibited CC-induced complement activation by inhibiting binding of the pattern recognition molecules C1q (via IgM) and ficolin-2. The reduced CC-induced complement activation mediated by α-cyclodextrin resulted in reduced phagocytosis and reduced ROS production in monocytes and granulocytes. Alpha-cyclodextrin was the most effective inhibitor of CC-induced complement activation, with the reduction in deposition of complement activation products being significantly different from the reduction induced by 2-hydroxypropyl-β-cyclodextrin. We also found that α-cyclodextrin was able to dissolve CCs. ConclusionsThis study identified α-cyclodextrin as a potential candidate in the search for therapeutics to prevent CC-induced inflammation in atherosclerosis.

Cancer‐associated fibroblasts promote M2 polarization of macrophages in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is characterized by remarkable desmoplasia with infiltration of distinct cellular components. Cancer‐associated fibroblasts (CAFs) has been shown to be among the most prominent cells and played a significant role in shaping the tumor microenvironment by interacting with other type of cells. Here, we aimed to investigate the effect of CAFs in modulating phenotype of tumor‐associated macrophages (TAM). Under treatment of CAFs conditioned medium (CM) or direct co‐culture with CAFs, monocytes exhibited enhanced expression of CD206 and CD163 compared with control group (P < 0.01). The induction of M2 polarization was mediated by increased reactive oxygen species (ROS) production in monocytes as ROS elimination abolished the effect of CAFs (P < 0.05). The supernatant analysis showed that pancreatic CAFs produced increased macrophage colony‐stimulating factor (M‐CSF). Upon treatment of M‐CSF neutralizing antibody, the ROS generation and M2 polarization of CAFs CM‐stimulated monocytes were significantly inhibited (P < 0.05). In addition, the CAFs‐induced M2 macrophages significantly enhanced pancreatic tumor cell growth, migration, and invasion. Collectively, our data revealed that pancreatic CAFs were able to induce a tumor‐promoting TAM phenotype partly through secreted M‐CSF and enhanced ROS production in monocytes, indicating possible treatment strategies by targeting stromal cell interaction within PDAC microenvironment.

Increased monocyte-derived reactive oxygen species in type 2 diabetes: role of endoplasmic reticulum stress.

What is the central question of this study? Patients with type 2 diabetes exhibit increased oxidative stress in peripheral blood mononuclear cells, including monocytes; however, the mechanisms remain unknown. What is the main finding and its importance? The main finding of this study is that factors contained within the plasma of patients with type 2 diabetes can contribute to increased oxidative stress in monocytes, making them more adherent to endothelial cells. We show that these effects are largely mediated by the interaction between endoplasmic reticulum stress and NADPH oxidase activity. Recent evidence suggests that exposure of human monocytes to glucolipotoxic media to mimic the composition of plasma of patients with type 2 diabetes (T2D) results in the induction of endoplasmic reticulum (ER) stress markers and formation of reactive oxygen species (ROS). The extent to which these findings translate to patients with T2D remains unclear. Thus, we first measured ROS (dihydroethidium fluorescence) in peripheral blood mononuclear cells (PBMCs) from whole blood of T2D patients (n=8) and compared the values with age-matched healthy control subjects (n=8). The T2D patients exhibited greater basal intracellular ROS (mean±SD, +3.4±1.4-fold; P<0.05) compared with control subjects. Next, the increase in ROS in PBMCs isolated from T2D patients was partly recapitulated in cultured human monocytes (THP-1 cells) exposed to plasma from T2D patients for 36h (+1.3±0.08-fold versus plasma from control subjects; P<0.05). In addition, we found that increased ROS formation in THP-1 cells treated with T2D plasma was NADPH oxidase derived and led to increased endothelial cell adhesion (+1.8±0.5-fold; P<0.05) and lipid uptake (+1.3±0.3-fold; P<0.05). Notably, we found that T2D plasma-induced monocyte ROS and downstream functional effects were abolished by treating cells with tauroursodeoxycholic acid, a chemical chaperone known to inhibit ER stress. Collectively, these data indicate that monocyte ROS production with T2D can be attributed, in part, to signals from the circulating environment. Furthermore, an interplay between ER stress and NADPH oxidase activity contributes to ROS production and may be a mechanism mediating endothelial cell adhesion and foam cell formation in T2D.

Luteolin and fisetin suppress oxidative stress by modulating sirtuins and forkhead box O3a expression under in vitro diabetic conditions.

BACKGROUND/OBJECTIVEChronic hyperglycemia induces oxidative stress via accumulation of reactive oxygen species (ROS) and contributes to diabetic complications. Hyperglycemia induces mitochondrial superoxide anion production through the increased activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. This study aimed to determine whether fisetin and luteolin treatments suppress the oxidative stress by modulating the expression of sirtuins (SIRTs) and forkhead box O3a (FOXO3a) under hyperglycemic conditions in human monocytes.MATERIALS/METHODSHuman monocytic cells (THP-1) were cultured under osmotic control (14.5 mmol/L mannitol), normoglycemic (NG, 5.5 mmol/L glucose), or hyperglycemic (HG, 20 mmol/L glucose) conditions, in the absence or presence of fisetin and luteolin for 48 h. To determine the effect of fisetin and luteolin treatments on high glucose-induced oxidative stress, western blotting and intracellular staining were performed.RESULTSHyperglycemic conditions increased the ROS production, as compared to normoglycemic condition. However, fisetin and luteolin treatments inhibited ROS production under hyperglycemia. To obtain further insight into ROS production in hyperglycemic conditions, evaluation of p47phox expression revealed that fisetin and luteolin treatments inhibited p47phox expression under hyperglycemic conditions. Conversely, the expression levels of SIRT1, SIRT3, SIRT6, and FOXO3a were decreased under high glucose conditions compared to normal glucose conditions, but exposure to fisetin and luteolin induced the expression of SIRT1, SIRT3, SIRT6, and FOXO3a. The above findings suggest that fisetin and luteolin inhibited high glucose-induced ROS production in monocytes through the activation of SIRTs and FOXO3a.CONCLUSIONSThe results of our study supports current researches that state fisetin and luteolin as potential agents for the development of novel strategies for diabetes.

Porphyromonas gingivalis promotes low-density lipoprotein oxidation and atherosclerosis

ObjectivesRecent studies have reported that periodontal disease increases the risk of atherosclerosis. We previously reported that Porphyromonas gingivalis (Pg) accelerated atherosclerotic plaque formation in hyperlipidemic apoE-/- mice by initiating inflammation. Because the oxidative modification of lipoprotein plays a major role in atherosclerosis, we characterized the reactive oxygen species (ROS) produced by Pg and its ability to oxidize low-density lipoprotein (LDL). MethodsAtherosclerotic plaque formation in the aortic sinuses of Apoeshl mice injected intravenously with Pg 381 was assessed by Oil Red O staining. Anti-mouse antibodies to HOCl-oxidized LDL and 4-HNE, PLA2, MPO, and CD36 were used for immunohistochemistry. Intracellular ROS detection was performed using 2′,7′-dichlorodihydrofluorescein diacetate. Quantitative reverse transcription polymerase chain reaction was performed using primers specific for TLR-2, TLR-4, TLR-9, NOD-1, LOX-1, NOX-2, NOX-4, p22phox, and p47phox. ResultsPg challenge significantly induced ox-LDL and 4-HNE-, PLA2-, MPO-, and CD36-positive areas in proximal aortic lesions. TLR-2, TLR-4, NOD-1, LOX-1, and NADPH oxidase subunit-specific mRNA levels in the aorta were significantly increased. Furthermore, Pg significantly induced ROS production in monocytes. ConclusionsThese results suggest that Pg promotes LDL oxidation and contributes to atheroma development.

Hyperketonemia induces upregulation of LFA-1 in monocytes, which is mediated by ROS and P38 MAPK activation

Type 1 diabetic patients have hyperketonemia, elevated levels of pro-inflammatory and oxidative stress markers, and a higher incidence of vascular disease. This study examines the hypothesis that hyperketonemia increases reactive oxygen species (ROS) and is in part responsible for increased expression of adhesion molecules in monocytes. THP-1 monocytes were treated with acetoacetate (AA) or β-hydroxybutyrate (BHB) (0-10mmol/L) for 24h. Results show that AA, but not BHB, increases ROS production in monocytes. Pretreatment of monocytes with N-acetylcysteine (NAC) inhibited AA-induced ROS production. AA treatment induced upregulation of LFA-1 and pretreatment of monocytes with NAC or an inhibitor to p38 MAPK inhibited this upregulation in monocytes. This suggests that physiological concentrations of AA can contribute to increased ROS and activation of p38 MAPK, which may be responsible for AA-induced upregulation of LFA-1 in monocytes. Thus, hyperketonemia contributes to the risk for cardiovascular disease in type 1 diabetes.

ERIODICTYOL DECREASES REACTIVE OXYGEN SPECIES PRODUCTION AND INHIBITS P47PHOXCLUSTERING AND TRANSLOCATION IN MONOCYTES

Flavonoids have been proposed to have anti-inflammatory activity. Reactive oxygen species (ROS) are important mediators in the inflammatory response. We examined the effects of luteolin, quercetin and eriodictyol on ROS production in phorbol-12-myristate-13-acetate (PMA)-stimulated U937 cells. Eriodictyol inhibited ROS production at a lower inhibition concentration 50% (IC50) then luteolin and quercetin (eriodictyol : 0.822 µM, quercetin : 1.134 µM, luteolin : 1.026 µM). Moreover, eriodictyol inhibited ROS production in a dose- and time-dependent manner. Moreover, the p47phox subunit of nicotinamide adenine dinucleotide phosphate oxidase regulated PMA-mediated ROS production. After eriodictyol treatment, p47phox clustering and translocation also decreased on stimulation of U937 cells with PMA. This study suggests that eriodictyol governs immune responses by modulating p47phox clustering and translocation and ROS production in monocytes – evidence that eriodictyol is an immune regulator of ROS production during inflammatory responses. PRACTICAL APPLICATIONS This study suggests that eriodictyol governs immune responses by modulating p47phox clustering and translocation and ROS production in monocytes – evidence that eriodictyol is an immune regulator of ROS production during inflammatory responses.

Curcumin enhances non-opsonic phagocytosis of Plasmodium falciparum through up-regulation of CD36 surface expression on monocytes/macrophages

Curcumin is a natural plant product with antimalarial activity and immunomodulatory properties. In this study we aimed to investigate its effects on CD36 expression and CD36-mediated Plasmodium falciparum phagocytosis as well as the role played by reactive oxygen species (ROS) and the peroxisome proliferator-activated receptor γ retinoid X receptor (PPARγ-RXR) in these processes. In vitro antimalarial activity was evaluated by the [³H]hypoxanthine assay. ROS production and surface CD36 in human monocyte/macrophages were measured by flow cytometry. PPARγ and CD36 mRNA expression was determined by the QuantiGene Plex® assay and RT-qPCR. Nuclear PPARγ activation was analysed by a DNA-binding ELISA while nuclear erythroid-related factor 2 (Nrf2) expression was analysed by western blotting. P. falciparum phagocytosis was assessed by light microscopy. Curcumin's antimalarial activity was confirmed and did not differ between drug-susceptible and -resistant P. falciparum strains. Curcumin increased monocyte ROS production and expression of PPARγ and CD36 at the mRNA and protein levels. Although PPARγ activation was blocked by the PPARγ antagonist GW9662, CD36 expression and CD36-mediated P. falciparum phagocytosis were only inhibited by N-acetylcysteine (NAC), suggesting a PPARγ-independent CD36 expression pathway. We then identified seven putative Nrf2 antioxidant response elements on the CD36 gene promoter and showed that NAC inhibited curcumin-induced Nrf2 protein expression. CD36 expression and CD36-mediated P. falciparum phagocytosis by curcumin are dependent on ROS production and probably involve the Nrf2 pathway. The dual immunomodulatory and antimalarial mechanisms of curcumin action may mean that curcumin has potential as an adjuvant treatment limiting the risk of recrudescence following standard antimalarial therapy.

Maitake Mushroom Extract in Myelodysplastic Syndrome (MDS): A Phase II Study

Abstract 5043 Background:MDS is a heterogeneous group of hematologic diseases characterized by ineffective erythropoiesis and an increased risk of AML. MDS-related deaths are commonly due to infection resulting from impaired immunity, including quantitative and functional neutrophil defects. Maitake extract is from the fruit body of Grifola frondosa mushroom and is widely used in Asia as an adjunctive treatment for malignant diseases. This hot water extract is composed of a central beta 1,6-glucan core surrounded by beta 1,3-branches. Maitake extract enhances hematopoiesis by increasing G-CSF production with subsequent stem cell proliferation and differentiation of granulocyte-monocyte colony forming cells. Maitake also activates macrophages through Dectin-1 receptor binding, increases the weight and number of nucleated cells in the spleen and improves innate immune response in animal and human models. Activated macrophages may reduce iron stores. Compared to G-CSF, maitake may have fewer side-effects, is less costly, and may produce a comparable improvement in granulocyte function. Materials and Methods:MDS patients with IPSS Low and Int-1 risk disease and ANC>500, plts >20K were enrolled. Following double baseline measurements, patients received the maitake extract at 3mg/kg PO BID for 12 weeks. Primary endpoints include change in neutrophil counts and alterations in granulocyte function as measured by the respiratory burst test. The respiratory burst test is a highly sensitive and quantitative flow cytometric assay of granulocyte and monocyte function to assess the production of spontaneous reactive oxygen species (ROS) and stimulated response to 3 activators: opsonized E. coli, phorbol ester and the chemotactic peptide N-formylmethiolyl-leucyl-phenylalanine (fMLP). Results were compared to age-matched normal healthy volunteers. Responses were assessed using the modified International Working Group (IWG) MDS response criteria.Secondary endpoints include serial assessment of hemoglobin and platelet levels, reticulocyte count, monocyte function, and GM-CSF and G-CSF levels. Iron studies were assessed throughout treatment to determine whether the maitake indirectly alters iron stores potentially decreasing iron overload. Results:14 eligible patients (pts) (8M, 6F; median age 69, range 52–83 yrs) received the maitake extract. IPSS risk categories included: Low-risk, n=7; Int-1, n=7. WHO Stage: RA=2; RARS=1; RCMD=6; RCMD-RS=1; RCUD=1; MDS-U=1; CMML-1=2. Two pts received prior 5-azacytidine therapy.Mean fMLP-stimulated granulocyte function in pts at baseline was lower than control values. Monocyte baseline response to E. coli was significantly reduced (p<0.01); ROS production to phorbol ester was also lower than in controls.No patients met criteria for response using the modified IWG response criteria. Preliminary analyses showed that ROS responses to E. coli stimulation increased by weeks 7–9 in pts who were deficient at baseline. Monocyte ROS production increased in 2 pts by 2–6 fold at week 7. There was a 2–15 fold increase in the neutrophil response to fMLP by week 7 and a 2–35 fold increase in monocyte response to fMLP at week 9. Monocyte response to E. coli increased between 3–10 fold in 2 pts with peak values during weeks 7–9. The two pts with the most pronounced response by fMLP and ROS production also had eosinophilia, which resolved following study participation.The most common toxicities were diarrhea (grade 1) in 6 pts, eosinophilia (2–2.8 × normal) in 3 pts, increases in AST (grade 1) and ALT (grade 1) in 4 pts and nausea (grade 1) in 1 patient. Two pts were removed from the study, one for diarrhea possibly related to study medication and one for disease progression. Conclusion:Maitake mushroom extract is well tolerated without evidence of clinically significant adverse hepatic, renal or metabolic abnormalities. No clinical responses were seen. Based on our preliminary data, the maitake extract appears to enhance ROS in both unstimulated and stimulated cells, confirming preclinical data. The improvement in granulocyte and monocyte function in our in vitro studies suggests that maitake may enhance immune responses in MDS pts. Eosinophilia was noted but the significance remains uncertain at this time. The study is ongoing. Updated results will be presented. Disclosures:No relevant conflicts of interest to declare.

Inhibitory effect of estrogen on Rac1-expression in monocytes

Recruitment of circulating monocytes into the vasculature and release of reactive oxygen species (ROS) promote atherogenesis. Rac1-GTPase is an essential component of the superoxide-producing NADPH-oxidase complex. Estrogens inhibit production of vascular reactive oxygen species.Angiotensin II as well as overexpression of the constitutively active mutant RacL61 increased ROS production in monocytes. AngII-mediated ROS release was completely inhibited by overexpression of the dominant negative mutant RacN17 or treatment with 17β-estradiol. 17β-Estradiol reduced Rac1-expression concentration- and time-dependently and decreased basal, as well as AngII-induced Rac1 activity. The effects of 17β-estradiol were receptor-mediated. In vivo, down-regulation of Rac1 by 17β-estradiol was observed in human mononuclear cells of women with elevated 17β-estradiol levels after controlled ovarian hyperstimulation.In summary, the data show that down-regulation of Rac1-GTPase contributes to the inhibition of angiotensin II-mediated superoxide release by 17β-estradiol in monocytes.

Role of exercise intensities in oxidized low-density lipoprotein-mediated redox status of monocyte in men

Exercise significantly influences the progression of atherosclerosis. Oxidized LDL (ox-LDL), as a stimulator of oxidative stress, facilitates monocyte-related atherogenesis. This study investigates how exercise intensity impacts ox-LDL-mediated redox status of monocytes. Twenty-five sedentary healthy men exercised mildly, moderately, and heavily (i.e., 40, 60, and 80% maximal oxygen consumption, respectively) on a bicycle ergometer. Reactive oxygen species (ROS) production, cytosolic and mitochondrial superoxide dismutase (c-SOD and m-SOD, respectively) activities, and total and reduced-form gamma-glutamylcysteinyl glycine (t-GSH and r-GSH, respectively) contents in monocytes mediated by ox-LDL were measured. This experiment obtained the following findings: 1) ox-LDL increased monocyte ROS production and was accompanied by decreased c-SOD and m-SOD activities, as well as t-GSH and r-GSH contents, whereas treating monocytes with diphenyleneiodonium (DPI) (a NADPH oxidase inhibitor) or rotenone/2-thenoyltrifluoroacetone (TTFA) (mitochondrial complex I/II inhibitors) hindered ox-LDL-induced monocyte ROS production; 2) production of ROS and reduction of m-SOD activity and r-GSH content in monocyte by ox-LDL were enhanced by heavy exercise and depressed by mild and moderate exercise; and 3) heavy exercise augmented the inhibition of ox-LDL-induced monocyte ROS production by DPI and rotenone/TTFA, whereas these DPI- and rotenone/TTFA-mediated monocyte ROS productions were unchanged in response to mild and moderate exercise. We conclude that heavy exercise increases ox-LDL-induced monocyte ROS production, possibly by decreasing m-SOD activity and r-GSH content in monocytes. However, mild and moderate exercise likely protects individuals against suppression of anti-oxidative capacity of monocyte by ox-LDL.

Leptin stimulates the oxidative burst in control monocytes but attenuates the oxidative burst in monocytes from HIV-infected patients

Leptin, the 16 kDa product of the ob gene, is a an adipocyte-secreted hormone that centrally regulates weight. However, the physiological role of leptin is not limited to the regulation of food intake and energy expenditure, and leptin has a variety of effects in peripheral tissues, such as a regulatory role modulating the immune system. Thus, leptin receptor is expressed in human peripheral blood mononuclear cells, mediating the leptin stimulation of proliferation and activation, the production of proinflammatory cytokines from cultured monocytes, and the prevention of apoptotic death in serum-deprived monocytes. Because leptin can stimulate monocytes and the production of reactive oxygen species (ROS) are the result of monocyte activation, we investigated the effect of leptin on ROS production by human monocytes in vitro. Oxidative burst was measured by oxidation of the redox-sensitive dye 2',7'-dichlorofluorescein diacetate, and analysed by flow cytometry. We have found that stimulation with leptin produces oxygen radical formation by monocytes. This effect is dependent on the dose and maximal response is achieved at 10 nM leptin. Because HIV infection induces the production of ROS, we next investigated the effect of leptin on ROS production in monocytes from HIV-positive (HIV+) subjects. We have also found that monocytes from HIV+ subjects spontaneously produced increased amounts of free radicals. In contrast, leptin stimulation of monocytes from these patients partially inhibited the production of ROS. This effect of leptin was also dependent on the dose and maximal effect was achieved at 10 nM. The effect of leptin stimulating the production of ROS is consistent with the proinflammatory role in the immune system. On the other hand, the inhibitory effect on monocytes from HIV+ subjects may be explained by the attenuation of the oxidative burst by a delayed activation of monocytes in a hyperinflammatory state.

Protective properties of artichoke (Cynara scolymus) against oxidative stress induced in cultured endothelial cells and monocytes

It is currently believed that oxidative stress and inflammation play a significant role in atherogenesis. Artichoke extract exhibits hypolipemic properties and contains numerous active substances with antioxidant properties in vitro. We have studied the influence of aqueous and ethanolic extracts from artichoke on intracellular oxidative stress stimulated by inflammatory mediators (TNFα and LPS) and ox-LDL in endothelial cells and monocytes. Oxidative stress which reflects the intracellular production of reactive oxygen species (ROS) was followed by measuring the oxidation of 2′, 7′-dichlorofluorescin (DCFH) to 2′, 7′-dichlorofluorescein (DCF). Agueous and ethanolic extracts from artichoke were found to inhibit basal and stimulated ROS production in endothelial cells and monocytes in dose dependent manner. In endothelial cells, the ethanolic extract (50 μg/ml) reduced ox-LDL-induced intracellular ROS production by 60% (p<0,001) while aqueous extract (50 μg/ml) by 43% (p<0,01). The ethanolic extract (50 μg/ml) reduced ox-LDL-induced intracellular ROS production in monocytes by 76% (p<0,01). Effective concentrations (25–100 μg/ml) were well below the cytotoxic levels of the extracts which started at 1 mg/ml as assessed by LDH leakage and trypan blue exclusion. Penetration of some active substances into the cells was necessary for inhibition to take place as juged from the effect of preincubation time. These results demonstrate that artichoke extracts have marked protective properties against oxidative stress induced by inflammatory mediators and ox-LDL in cultured endothelial cells and monocytes.

Aminoethyl-isothiourea, a nitric oxide synthase inhibitor and oxygen radical scavenger, improves survival and counteracts hemodynamic deterioration in a porcine model of streptococcal shock.

To test the effect of a continuous infusion of the nitric oxide (NO) synthase (S) inhibitor aminoethyl-isothiourea (AE-ITU) on survival time, hemodynamics, and oxygen transport in a porcine model of live group A streptococcal (GAS) sepsis. Furthermore, to examine the role of endothelin-1, histamine, and reactive oxygen species (ROS) in streptococcal shock. Prospective, randomized trial. Laboratory at a university hospital. Twenty-eight pigs with an average weight of 25 kg. Sixteen animals received a continuous infusion of live Streptococcus pyogenes 1.3 x 10(10) colony forming units/hr: eight received fluids only, and the other eight received an intravenous infusion of AE-ITU 10 mg/kg/hr starting 30 mins before the GAS challenge. Six control pigs received AE-ITU 10 mg/kg/hr iv for 5 hrs. Another six animals received half the dose of GAS over 5 hrs. GAS infusion caused a rapid increase in pulmonary, hepatic, and systemic vascular resistance, followed by hypotension with a 90% lethality at 4 hrs. Treatment with AE-ITU increased 4-hr survival in septic animals from 1/8 to 8/8 and 5-hr survival from 0/8 to 5/8, prevented hypotension, and increased urine output. AE-ITU attenuated the decrease in cardiac output, liver blood flow, and oxygen delivery, and hepatic arterial blood flow as a fraction of cardiac output increased (all p < .05). Plasma nitrate/nitrite levels decreased in all animals. Inducible NOS and endothelial constitutive NOS activities in liver, gut, and lung were not increased during sepsis, nor were they decreased after AE-ITU. Plasma levels of endothelin-1 and methylhistamine increased in all septic animals and were not modified by AE-ITU. AE-ITU prevented the increase in monocyte ROS production caused by GAS. In control animals, AE-ITU caused an increase in mean arterial pressure, liver blood flow, and oxygen delivery. In this model of porcine GAS-induced septic shock, which was not associated with enhanced NO production, infusion of the NOS inhibitor AE-ITU prolonged survival, prevented hypotension, and improved cardiac contractility, organ perfusion, and tissue oxygenation. These beneficial effects of AE-ITU might be a result of the combined effect of ROS scavenging and modulation of local NO production, thus improving the balance of vasodilator and vasoconstrictor forces and reducing oxidative stress.