Lower Reactive Oxygen Species Production Research Articles

Clinical and Genomic Correlates of Neutrophil Reactive Oxygen Species Production in Pediatric Patients With Crohn’s Disease

Individuals with monogenic disorders of phagocyte function develop chronic colitis that resembles Crohn's disease (CD). We tested for associations between mutations in genes encoding reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, neutrophil function, and phenotypes of CD in pediatric patients. We performed whole-exome sequence analysis to identify mutations in genes encoding NADPH oxidases (such as CYBA, CYBB, NCF1, NCF2, NCF4, RAC1, and RAC2) using DNA from 543 pediatric patients with inflammatory bowel diseases. Blood samples were collected from an additional 129 pediatric patients with CD and 26 children without IBD (controls); we performed assays for neutrophil activation, reactive oxygen species (ROS) production, and bacteria uptake and killing. Whole-exome sequence analysis was performed using DNA from 46 of the children with CD to examine associations with NADPH gene mutations; RNA sequence analyses were performed using blood cells from 46 children with CD to test for variations in neutrophil gene expression associated with ROS production. We identified 26 missense mutations in CYBA, CYBB, NCF1, NCF2, and NCF4. Patients with CD who carried mutations in these genes were 3-fold more likely to have perianal disease (P= .0008) and stricturing complications (P= .002) than children with CD without these mutations. Among patients withCD with none of these mutations, 9% had undergone abdominal surgery; among patients with mutations in these NADPH oxidase genes, 31% had undergone abdominal surgery (P=.0004). A higher proportion of neutrophils from children with CD had low ROS production (47%) than from controls (15%) among the 129 patients tested for ROS (P= .002). Minor alleles of the NADPH genes were detected in 7% of children with CD whose neutrophils produced normal levels of ROS vs 38% of children whose neutrophils produced low levels of ROS (P= .009). Neutrophils that produced low levels of ROS had specific alterations in genes that regulate glucose metabolism and antimicrobial responses. We identified missense mutations in genes that encode NADPH oxidases in children with CD; these were associated with a more aggressive disease course and reduced ROS production by neutrophils from the patients.

Plant cell suspension culture of Thevetia peruviana (Pers.) K. Schum. in shake flask and stirred tank reactor scale: A comparative study

The aim of this work was to evaluate the effect of changing the shake flask for a stirred-tank bench bioreactor on the performance of Thevetia peruviana cultures. Maximum cell growth of 18.59 and 8.46 g DW/L for shake flask and bench bioreactor at day 14 was obtained, respectively. The end of the exponential growth phase in shake flask was correlated with total sugar consumption, while this behavior was not observed in bench bioreactor. Preferential uptake of glucose over fructose was observed in both systems. Cell morphology was similar for both systems during culture time, exhibiting individual cells with cylindrical and elongated shape at day 0 and some aggregates with rounded cells at day 14. Polyphenol production was not affected by the system configuration and scale. Intra and extracellular antioxidant activity were directly related with phenol production in both systems. Guaiacol peroxidase activity (GPX) went from 1.87 to 7.97 µM/min/mg protein in shake flask and it correlated with low reactive oxygen species (ROS) production. This is an indication of a positive enzymatic cell response for culture in shake flask mediated by GPX. In contrast, the conditions in the bench bioreactor generated a higher stress environment, increasing ROS production without activating enzymatic or non-enzymatic responses led by guaiacol and phenolic compounds, respectively. In conclusion, bench stirred-tank bioreactor scale affects biomass production and sugars uptake negatively. Key words: Thevetia peruviana, plant cell suspension culture, stirred-tank bioreactor, phenolic compounds, antioxidant activity.

Impact of Bariatric Surgical Intervention on Peripheral Blood Neutrophil (PBN) Function in Obesity

AimThe aim of this study was to investigate the impact of weight loss following gastric band surgery on multiple measures of peripheral blood neutrophil (PBN) function.Material and MethodsTwenty-three obese patients undergoing gastric band surgery were recruited to a longitudinal intervention study, alongside non-obese, healthy gender- and age-matched controls. Eighteen pairs of patients and controls completed all stages of the study. PBNs were isolated by density centrifugation and a comprehensive analysis of PBN function was undertaken at various stages of the patients’ bariatric surgical care pathway.ResultsObese patients exhibited exaggerated PBN activity in response to various stimuli, characterised by higher reactive oxygen species (ROS) generation (n = 18, p < 0.001) and release of pro-inflammatory cytokines (n = 10, p < 0.05) and lower PBN extracellular trap (NET) formation (n = 18, p < 0.01). PBN chemotactic accuracy was also impaired prior to surgery (n = 18, p < 0.01). Weight loss was associated with normalised NET production and lower ROS production and cytokine release relative to healthy controls. However, chemotactic accuracy remained impaired in patients.ConclusionsWeight loss following gastric band surgery was associated with a decrease in the pro-inflammatory activities of peripheral blood neutrophils (PBNs). A hyper-inflammatory PBN phenotype, involving excess ROS and cytokine release, reduced NET formation and chemotaxis, may lead to a reduced ability to eliminate infection, alongside inflammation-mediated tissue damage in obese individuals.

Au Nanoclusters Sensitized Black TiO2-x Nanotubes for Enhanced Photodynamic Therapy Driven by Near-Infrared Light.

The low reactive oxygen species production capability and the shallow tissue penetration of excited light (UV) are still two barriers in photodynamic therapy (PDT). Here, Au cluster anchored black anatase TiO2-x nanotubes (abbreviated as Au25 /B-TiO2-x NTs) are synthesized by gaseous reduction of anatase TiO2 NTs and subsequent deposition of noble metal. The Au25 /B-TiO2-x NTs with thickness of about 2 nm exhibit excellent PDT performance. The reduction process increased the density of Ti3+ on the surface of TiO2 , which effectively depresses the recombination of electron and hole. Furthermore, after modification of Au25 nanoclusters, the PDT efficiency is further enhanced owing to the changed electrical distribution in the composite, which forms a shallow potential well on the metal-TiO2 interface to further hamper the recombination of electron and hole. Especially, the reduction of anatase TiO2 can expend the light response range (UV) of TiO2 to the visible and even near infrared (NIR) light region with high tissue penetration depth. When excited by NIR light, the nanoplatform shows markedly improved therapeutic efficacy attributed to the photocatalytic synergistic effect, and promotes separation or restrained recombination of electron and hole, which is verified by experimental results in vitro and in vivo.

Prostacyclin mimetics afford protection against lipopolysaccharide/d-galactosamine-induced acute liver injury in mice

Prostacyclin (PGI2) serves as a protective, anti-inflammatory mediator and PGI2 mimetics may be useful as a hepatoprotective agent. We examined whether two PGI2 mimetics, ONO-1301 and beraprost, are beneficial in acute liver injury and attempted to delineate the possible mechanism underlying the hepatoprotective effect. Acute liver injury was induced by lipopolysaccharide/d-galactosamine (LPS/GalN) in mice. Mice were given an intraperitoneal injection of PGI2 mimetics 1h before LPS/GalN challenge. Both ONO-1301 and beraprost significantly declined the LPS/GalN-induced increase in serum aminotransferase activity. ONO-1301 and, to a lesser extent, beraprost inhibited hepatic gene expression levels of pro-inflammatory cytokines, which were sharply elevated by LPS/GalN. The hepatoprotective effects of ONO-1301, to a lesser extent, of beraprost were also supported by liver histopathological examinations. The PGI2 receptor antagonist CAY10441 abrogated their hepatoprotective effects. The mechanisms behind the benefit of PGI2 mimetics in reducing LPS/GalN-induced liver injury involved, in part, their suppressive effects on increased generation of reactive oxygen species (ROS), since their ability to prevent LPS/GalN-induced hepatic apoptosis was mimicked by the antioxidant N-acetyl-l-cysteine. They significantly diminished LPS/GalN-induced activation of signal transducers and activators of transcription 3 (STAT3) in liver tissues, an effect which was highly associated with their hepatoprotective effects. We indicate that IP receptor activation with PGI2 mimetics can rescue the damage in the liver induced by LPS/GalN by undermining activation of STAT3 and leading to a lower production of ROS. Our findings point to PGI2 mimetics, especially ONO-1301, as a potential novel therapeutic modality for the treatment of acute liver injury.

Changes in mitochondrial properties may contribute to enhanced resistance to ischemia-reperfusion injury in the diabetic rat heart.

Diabetes mellitus, besides having deleterious effects, induces cardiac adaptation that may reduce the heart's susceptibility to ischemia-reperfusion (IR) injury. This study aimed to investigate whether changes in mitochondrial properties are involved in the mechanisms of increased resistance of the diabetic heart to IR. Adult male Wistar rats were made diabetic by a single dose of streptozotocin (65 mg·kg-1, i.p.), and on the day 8, Langendorff-perfused hearts were subjected to 30 min global ischemia and 40 min reperfusion. Baseline preischemic parameters in the diabetic hearts did not differ markedly from those in the nondiabetic controls, except for lower left ventricular developed pressure, higher mitochondrial membrane fluidity, and protein levels of manganese superoxide dismutase. On the other hand, diabetic hearts showed significantly better post-IR functional restoration and reduced arrhythmogenesis associated with lower reactive oxygen species production as compared with healthy controls. IR decreased membrane fluidity in both experimental groups; however, it led to a complete recovery of mitochondrial Mg2+-ATPase activity in diabetics in contrast to its reduction in nondiabetics. These findings indicate that the heart may become adapted to diabetes-induced alterations that might increase its tolerance to an ischemic insult. Preserved mitochondrial function might play a role in the mechanisms of the heart's resistance to IR injury in diabetics.

Modulation of oxidative phosphorylation and redox homeostasis in mitochondrial NDUFS4 deficiency via mesenchymal stem cells

BackgroundDisorders of the oxidative phosphorylation (OXPHOS) system represent a large group among the inborn errors of metabolism. The most frequently observed biochemical defect is isolated deficiency of mitochondrial complex I (CI). No effective treatment strategies for CI deficiency are so far available. The purpose of this study was to investigate whether and how mesenchymal stem cells (MSCs) are able to modulate metabolic function in fibroblast cell models of CI deficiency.MethodsWe used human and murine fibroblasts with a defect in the nuclear DNA encoded NDUFS4 subunit of CI. Fibroblasts were co-cultured with MSCs under different stress conditions and intercellular mitochondrial transfer was assessed by flow cytometry and fluorescence microscopy. Reactive oxygen species (ROS) levels were measured using MitoSOX-Red. Protein levels of CI were analysed by blue native polyacrylamide gel electrophoresis (BN-PAGE).ResultsDirect cellular interactions and mitochondrial transfer between MSCs and human as well as mouse fibroblast cell lines were demonstrated. Mitochondrial transfer was visible in 13.2% and 6% of fibroblasts (e.g. fibroblasts containing MSC mitochondria) for human and mouse cell lines, respectively. The transfer rate could be further stimulated via treatment of cells with TNF-α. MSCs effectively lowered cellular ROS production in NDUFS4-deficient fibroblast cell lines (either directly via co-culture or indirectly via incubation of cell lines with cell-free MSC supernatant). However, CI protein expression and activity were not rescued by MSC treatment.ConclusionThis study demonstrates the interplay between MSCs and fibroblast cell models of isolated CI deficiency including transfer of mitochondria as well as modulation of cellular ROS levels. Further exploration of these cellular interactions might help to develop MSC-based treatment strategies for human CI deficiency.

Silymarin inhibits adipogenesis in the adipocytes in grass carp Ctenopharyngodon idellus in vitro and in vivo.

In this study, two experiments were performed to explore the function of silymarin in adipogenesis in grass carp (Ctenopharyngodon idellus) using in vitro and in vivo models. In experiment 1, differentiated grass carp pre-adipocytes were treated with silymarin for 6days. Treatment with 100μgmL-1silymarin (SM100 group) significantly reduced triglyceride accumulation at day 6. The adipogenic gene expression levels of PPARγ, C/EBPα, SREBP1c, FAS, SCD1, and LPL, and the protein expression level of PPARγ were significantly down-regulated in the SM100 group. Additionally, the SM100 group had significantly lower reactive oxygen species production and reduced glutathione contents compared with the control in vitro. In experiment 2, the juvenile grass carp (mean body weight= 27.4±0.17g) were fed six isonitrogenous and isocaloric diets in a factorial design containing 0, 100, or 200mgkg-1 silymarin (SM0, SM100, SM200) associated with either 4 or 8% lipid levels (low lipid, LL, and high lipid, HL, respectively) for 82days. The results demonstrated that dietary silymarin supplementation significantly reduced the elevated intraperitoneal fat index in grass carp fed with high-lipid diets, and the gene expression of adipogenesis (PPARγ, FAS) when supplemented with dietary silymarin was notably lower than when no silymarin was supplemented under the high-lipid diets. Thus, our data suggest that silymarin suppressed lipid accumulation in grass carp both in vitro and in vivo, and the effect might be due to an influence on the expression of adipogenesis factors and ROS production partly associated with effects on antioxidant capability.

Evaluation of silicon influence on the mitigation of cadmium-stress in the development of Arabidopsis thaliana through total metal content, proteomic and enzymatic approaches

The mitigation of Cd-stress through Si addition to Arabidopsis thaliana cultivation is evaluated in terms of total metal content, proteomic and enzymatic approaches. Four different treatment are evaluated: TC (control, without Si or Cd addition), T1 (with Si addition), T2 (with Cd addition), and T3 (with Si and Cd addition). Through the total determination of Cd and Si in Arabidopsis leaves, the Cd concentration decreased by half when T2 is compared with T3 treatment. In terms of proteomic approach, some differential protein species are achieved by comparative proteomics through 2-D DIGE of all treatments evaluated. Fifty six differential abundant proteins spots (abundance factor ≥1.5) are detected, and 32 of them accurately characterized and identified through nESI-LC–MS/MS. These proteins are differentially produced due to Cd and/or Si treatments, which mainly include proteins associated with disease/defense, energy and metabolism. The most difference in the abundance of proteins is found due to the presence or absence of Si in plants treated with Cd. Regarding the enzymatic approaches, a major increase is found on APX, CAT and GR activities (5.0, 3.5, and 1.5-fold, respectively). The same is observed for the MDA concentration because an increase of 3-fold is found when TC are compared to those treated with T2. However, when T3 plants are evaluated, the enzymes activities are similar to TC plants. Differences ranging from 6.5 to 21% are detected considering the activity of SOD in the treatments (T1-T3 x TC). The decreased activities of CAT, APX and GR and lower MDA concentration indicate a lower reactive oxygen species production in plants treated with Cd and Si. Based on a proteomics point of view it is possible to conclude that Si-Cd interactions occur at protein level and allow plants to respond effectively to the Cd toxicity, revealing the active involvement of Si on mechanisms involved in Si-induced Cd tolerance in Arabidopsis plants. Additionally, from an enzymatic point of view, it is possible to conclude that Si positively interferes diminishing the negative effects of Cd in Arabidopsis by decreasing the reactive oxygen species generation and increasing the antioxidative enzyme activity.

Neonatal myeloid derived suppressor cells show reduced apoptosis and immunosuppressive activity upon infection with Escherichia coli.

Susceptibility to infection during the neonatal period and reduced control of inflammation in neonates are attributed to immunosuppression persisting from fetal life. Myeloid-derived suppressor cells (MDSCs) are immature myeloid progenitors with suppressive activity and increased numbers in cord blood. We hypothesized that MDSCs contribute to innate host defence in neonates, paralleled by anti-inflammatory signalling.Phagocytic activity, infection induced apoptosis, expression of B-cell lymphoma (Bcl)-2 family proteins, production of reactive oxygen species (ROS), cytokine production and T-cell suppression of neonatal granulocytic-MDSCs (G-MDSCs) after infection with Escherichia coli (E. coli) were compared to neonatal autologous mature polymorphonuclear leukocytes (PMNs). Phagocytic activity of G-MDSCs upon infection with E. coli was equal to that of mature PMNs, however, apoptosis of G-MDSCs was decreased. G-MDSCs showed enhanced Bcl-2-expression and lower ROS production compared to PMNs. Inhibition of Bcl-2 reduced apoptosis rates of G-MDSCs to that of mature PMNs. Induction of anti-inflammatory transforming growth factor beta (TGF-β) was enhanced, while pro-inflammatory IL-8 decreased in G-MDSCs compared to PMNs. Infected G-MDSCs strongly suppressed proliferation of T cells. We show a direct role of G-MDSCs for anti-bacterial host defence. Prolonged survival and anti-inflammatory capacity suggest that G-MDSCs are important for immune-regulation after bacterial infection.

Athero-inflammatory nanotherapeutics: Ferulic acid-based poly(anhydride-ester) nanoparticles attenuate foam cell formation by regulating macrophage lipogenesis and reactive oxygen species generation.

Future development of anti-oxidant formulations for atherosclerosis applications is essential to deliver an efficacious dose while limiting localized concentrations of pro-oxidants. In this study, we illustrate the potential of degradable ferulic acid-based polymer nanoparticles to control macrophage foam cell formation by significantly reducing oxLDL uptake through downregulation of scavenger receptors, CD-36, MSR-1, and LOX-1. Another critical finding is the ability of the degradable ferulate-based polymer nanoparticles to lower macrophage reactive oxygen species (ROS) levels, a precursor to apoptosis and plaque escalation. The degradable ferulic acid-based polymer nanoparticles hold significant promise as a means to alter the treatment and progression of atherosclerosis.

Examination of the superoxide/hydrogen peroxide forming and quenching potential of mouse liver mitochondria

Pyruvate dehydrogenase (PDHC) and α-ketoglutarate dehydrogenase complex (KGDHC) are important sources of reactive oxygen species (ROS). In addition, it has been found that mitochondria can also serve as sinks for cellular hydrogen peroxide (H2O2). However, the ROS forming and quenching capacity of liver mitochondria has never been thoroughly examined. Here, we show that mouse liver mitochondria use catalase, glutathione (GSH), and peroxiredoxin (PRX) systems to quench ROS. Incubation of mitochondria with catalase inhibitor 3-amino-1,2,4-triazole (triazole) induced a significant increase in pyruvate or α-ketoglutarate driven O2−/H2O2 formation. 1-Choro-2,4-dinitrobenzene (CDNB), which depletes glutathione (GSH), elicited a similar effect. Auranofin (AF), a thioredoxin reductase-2 (TR2) inhibitor which disables the PRX system, did not significantly change O2−/H2O2 formation. By contrast catalase, GSH, and PRX were all required to scavenging extramitochondrial H2O2. In this study, the ROS forming potential of PDHC, KGDHC, Complex I, and Complex III was also profiled. Titration of mitochondria with 3-methyl-2-oxovaleric acid (KMV), a specific inhibitor for O2−/H2O2 production by KGDHC, induced a ~86% and ~84% decrease in ROS production during α-ketoglutarate and pyruvate oxidation. Titration of myxothiazol, a Complex III inhibitor, decreased O2−/H2O2 formation by ~45%. Rotenone also lowered ROS production in mitochondria metabolizing pyruvate or α-ketoglutarate indicating that Complex I does not contribute to ROS production during forward electron transfer from NADH. Taken together, our results indicate that KGDHC and Complex III are high capacity sites for O2−/H2O2 production in mouse liver mitochondria. We also confirm that catalase plays a role in quenching either exogenous or intramitochondrial H2O2.

Pathogenetic role of endothelial nitric oxide synthase uncoupling during lung ischaemia-reperfusion injury.

Ischaemia-reperfusion injury is a necessary part of organ transplantation and a key determinant of both acute and chronic graft failure. We have assessed the contribution of endothelial nitric oxide synthase (eNOS) and eNOS uncoupling to oxidative and nitrosative stress formation during lung ischaemia-reperfusion injury dependent on ischaemia time. Forty eNOS wild-type mice (eNOS +/+ ) and 40 eNOS knock-out mice (eNOS -/- ) received either a sham thoracotomy or 60 or 90 min of ischaemia, followed by 0, 1 or 24 h of reperfusion. Lung tissue was analysed with electron spin resonance for NO production and reactive oxygen species content. Protein nitrosation, eNOS and eNOS uncoupling were determined using western blotting. In peripheral blood, arterial blood gases were taken and reactive oxygen species content was determined. eNOS +/+ mice had lower reactive oxygen species production in their peripheral circulation but worse blood gas values after 1 h of reperfusion. Lung tissue of eNOS -/- mice showed lower reactive oxygen species and NO production and lower protein nitrosation compared with wild-type mice. Longer ischaemia times result in more elaborate oxidative and nitrosative stress dependent on eNOS genotype. Structural eNOS uncoupling was present after 60 min of ischaemia but diminished after 90 min of ischaemia. eNOS uncoupling may contribute to lung ischaemia-reperfusion injury and inflammation. This ultimately leads to worse clinical outcome. Stabilizing eNOS may therefore be a new approach to extend pulmonary graft survival.

Metabolic analysis of radioresistant medulloblastoma stem-like clones and potential therapeutic targets.

Medulloblastoma is a fatal brain tumor in children, primarily due to the presence of treatment-resistant medulloblastoma stem cells. The energy metabolic pathway is a potential target of cancer therapy because it is often different between cancer cells and normal cells. However, the metabolic properties of medulloblastoma stem cells, and whether specific metabolic pathways are essential for sustaining their stem cell-like phenotype and radioresistance, remain unclear. We have established radioresistant medulloblastoma stem-like clones (rMSLCs) by irradiation of the human medulloblastoma cell line ONS-76. Here, we assessed reactive oxygen species (ROS) production, mitochondria function, oxygen consumption rate (OCR), energy state, and metabolites of glycolysis and tricarboxylic acid cycle in rMSLCs and parental cells. rMSLCs showed higher lactate production and lower oxygen consumption rate than parental cells. Additionally, rMSLCs had low mitochondria mass, low endogenous ROS production, and existed in a low-energy state. Treatment with the metabolic modifier dichloroacetate (DCA) resulted in mitochondria dysfunction, glycolysis inhibition, elongated mitochondria morphology, and increased ROS production. DCA also increased radiosensitivity by suppression of the DNA repair capacity through nuclear oxidization and accelerated the generation of acetyl CoA to compensate for the lack of ATP. Moreover, treatment with DCA decreased cancer stem cell-like characters (e.g., CD133 positivity and sphere-forming ability) in rMSLCs. Together, our findings provide insights into the specific metabolism of rMSLCs and illuminate potential metabolic targets that might be exploited for therapeutic benefit in medulloblastoma.

Tu1748 - Low Neutrophil Reactive Oxygen Species Production is Associated with Nicotinamide-Adenine Dinucleotide Phosphate (NADPH) Oxidase Gene Mutations and Refractory Colonic Involvement in Pediatric Crohn Disease

Tu1748 - Low Neutrophil Reactive Oxygen Species Production is Associated with Nicotinamide-Adenine Dinucleotide Phosphate (NADPH) Oxidase Gene Mutations and Refractory Colonic Involvement in Pediatric Crohn Disease

3-bromopyruvate and buthionine sulfoximine effectively kill anoikis-resistant hepatocellular carcinoma cells.

Background & aimsAcquisition of anoikis resistance is a prerequisite for metastasis in hepatocellular carcinoma (HCC). However, little is known about how energy metabolism and antioxidant systems are altered in anoikis-resistant (AR) HCC cells. We evaluated anti-tumor effects of a combination treatment of 3-bromopyruvate (3-BP) and buthionine sulfoximine (BSO) in AR HCC cells.MethodsWe compared glycolysis, reactive oxygen species (ROS) production, and chemoresistance among Huh-BAT, HepG2 HCC cells, and the corresponding AR cells. Expression of hexokinase II, gamma-glutamylcysteine synthetase (rGCS), and epithelial–mesenchymal transition (EMT) markers in AR cells was assessed. Anti-tumor effects of a combination treatment of 3-BP and BSO were evaluated in AR cells and an HCC xenograft mouse model.ResultsAR HCC cells showed significantly higher chemoresistance, glycolysis and lower ROS production than attached cells. Expression of hexokinase II, rGCS, and EMT markers was higher in AR HCC cells than attached cells. A combination treatment of 3-BP/BSO effectively suppressed proliferation of AR HCC cells through apoptosis by blocking glycolysis and enhancing ROS levels. In xenograft mouse models, tumor growth derived from AR HCC cells was significantly suppressed in the group treated with 3-BP/BSO compared to the group treated with 3-BP or sorafenib.ConclusionsThese results demonstrated that a combination treatment of 3-BP/BSO had a synergistic anti-tumor effect in an AR HCC model. This strategy may be an effective adjuvant therapy for patients with sorafenib-resistant HCC.

Insulin-like growth factor 1 receptor-mediated cell survival in hypoxia depends on the promotion of autophagy via suppression of the PI3K/Akt/mTOR signaling pathway.

Hypoxia is widely accepted as a fundamental biological phenomenon, which is strongly associated with tissue damage and cell viability under stress conditions. Insulin-like growth factor-1 (IGF-1) is known to protect tissues from multiple types of damage, and protect cells from apoptosis. Hypoxia is a regulatory factor of the IGF system, however the role of the IGF-1 receptor (IGF-1R) in hypoxia-induced apoptosis remains unclear. The present study investigated the potential mechanisms associated with IGF-1R-associated apoptosis under hypoxic conditions. Mouse embryonic fibroblasts exhibiting disruption or overexpression of IGF-1R (R- cells and R+ cells) were used to examine the level of apoptosis, autophagy, and production of reactive oxygen species (ROS). The autophagy inhibitor 3-methyladenine was used to assess the effect of autophagy on ROS production and apoptosis under hypoxic conditions. A potential downstream signaling pathway involving phosphatidylinositol 3-kinase (PI3K)/threonine protein kinase B (Akt)/mammalian target of rapamycin (mTOR) was identifiedby western blot analysis. The results demonstrated that hypoxia induced apoptosis, increased ROS production, and promoted autophagy in a time-dependent manner relative to that observed under normoxia. R+ cells exhibited a lower percentage of apoptotic cells, lower ROS production, and higher levels of autophagy when compared to that of R- cells. In addition, inhibition of autophagy led to increased ROS production and a higher percentage of apoptotic cells in the two cell types. Furthermore, IGF-1R is related with PI3K/Akt/mTOR signaling pathway and enhanced autophagy-associated protein expression, which was verified following treatment with the PI3K inhibitor LY294002. These results indicated that IGF-1R may increase cell viability under hypoxic conditions by promoting autophagy and scavenging ROS production, which is closed with PI3K/Akt/mTOR signaling pathway.

Specific pathways mediating inflammasome activation by Candida parapsilosis

Candida albicans and C. parapsilosis are human pathogens causing severe infections. The NLRP3 inflammasome plays a crucial role in host defence against C. albicans, but it has been previously unknown whether C. parapsilosis activates this complex. Here we show that C. parapsilosis induces caspase-1 activation and interleukin-1β (IL-1β) secretion in THP-1, as well as primary, human macrophages. IL-1β secretion was dependent on NLRP3, K+-efflux, TLR4, IRAK, Syk, caspase-1, caspase-8 and NADPH-oxidase. Importantly, while C. albicans induced robust IL-1β release after 4 h, C. parapsilosis was not able to stimulate the production of IL-1β after this short incubation period. We also found that C. parapsilosis was phagocytosed to a lesser extent, and induced significantly lower ROS production and lysosomal cathepsin B release compared to C. albicans, suggesting that the low extent of inflammasome activation by C. parapsilosis may result from a delay in the so-called “signal 2”. In conclusion, this is the first study to examine the molecular pathways responsible for the IL-1β production in response to a non-albicans Candida species, and these results enhance our understanding about the immune response against C. parapsilosis.

Differential reactive oxygen species production of neutrophils and their oxidative damage in patients with active and inactive systemic lupus erythematosus

ObjectiveIncreasing interest is given to the involvement of the innate immunity and especially Polymorphonuclear neutrophils (PMN) in the physiopathological process of inflammatory diseases such as systemic lupus erythematosus (SLE). Here, we investigated the oxidative burst and damages in SLE patients neutrophils, considering the two phases of the disease, the active and the remission/inactive states. MethodsThis study was conducted on 30 SLE patients and 23 healthy controls. The oxidative burst in neutrophils of SLE patients and controls was triggered by fMLP and TPA, while reactive oxygen species (ROS) production was evaluated using a chemiluminescence assay. Oxidative damages in neutrophils were assessed by measuring Free thiol groups level and carbonyl groups, as protein oxidative markers. The malondialdehyde (MDA) level informed about the lipid peroxidation (LPO) and the catalase activity indicated the antioxidant enzymatic activity. ResultCompared to controls, SLE patients exhibited a significantly increased level of ROS production concomitantly to a decreased response time. Their Neutrophils were characterized by a decreased level of MDA and high levels of protein oxidation as evidenced by increased carbonyl groups and decreased SH levels. The catalase activity was higher in SLE patients' neutrophils compared to controls. When patients were clustered according to the disease activity, PMN of patients in active phase showed, paradoxically, a lower ROS production and exhibited higher oxidative damages than the inactive group. ConclusionOur results highlight an altered behavior of LES patients derived PMN particularly in the active phase of the disease. The evaluation of the redox status including the rate of ROS production could be a biological marker to follow the activity of the disease.

PGC1α Activators Mitigate Diabetic Tubulopathy by Improving Mitochondrial Dynamics and Quality Control.

Purpose. In this study, we investigated the effect of PGC1α activators on mitochondrial fusion, fission, and autophagic quality control in renal tubular cells in a diabetic environment in vivo and in vitro. We also examined whether the upregulation of PGC1α attenuates diabetic tubulopathy by normalizing mitochondrial homeostasis. Methods. HKC8 cells were subjected to high-glucose conditions (30 mM D-glucose). Diabetes was induced with streptozotocin (STZ, 50 mg/kg i.p. for 5 days) in male C57/BL6J mice. AICAR or metformin was used as a PGC1α activator. Results. Treatment with the PGC1α activators AICAR and metformin improved functional mitochondrial mass in HKC8 cells in high-glucose conditions. Moreover, in renal proximal tubular cells, increased PGC1α activity correlated with the reversal of changes in Drp1, Mfn1, and LC3-II protein expression in a high-glucose environment. Normalized mitochondrial life cycles resulted in low ROS production and reduced apoptosis. AICAR and metformin treatment effectively mitigated albuminuria and renal histopathology and decreased the expression of TGFβ1 and αSMA in the kidneys of diabetic mice. Conclusions. Our results demonstrate that increases in PGC1α activity improve diabetic tubulopathy by modulating mitochondrial dynamics and autophagy.