Oxidation of Erythrocytes Enhance the Production of Reactive Species in the Presence of Artemisinins.

In red blood cells, hemoglobin iron represents the most plausible candidate to catalyze artemisinin activation but the limited reactivity of iron bound to hemoglobin does not play in favor for its direct involvement. Denatured hemoglobin appears a more likely candidate for artemisinin redox activation because it is expected to contain reactive iron and it has been described to release free heme and/or iron in erythrocyte. The aim of our study is to investigate, using three different methods: fluorescence, electron paramagnetic resonance and liquid chromatography coupled to mass spectrometry, how increasing the level of accessible iron into the red blood cells can enhance the reactive oxygen species (ROS) production derived from artemisinin. The over-increase of iron was achieved using phenylhydrazine, a strong oxidant that causes oxidative stress within erythrocytes, resulting in oxidation of oxyhemoglobin and leading to the formation of methemoglobin, which is subsequently converted into irreversible hemichromes (iron (III) compounds). Our findings confirmed, using the iron III chelator, desferrioxamine, the indirect participation of iron (III) compounds in the activation process of artemisinins. Furthermore, in strong reducing conditions, the activation of artemisinin and the consequent production of ROS was enhanced. In conclusion, we demonstrate, through the measurement of intra-erythrocytic superoxide and hydrogen peroxide production using various methods, that artemisinin activation can be drastically enhanced by pre-oxidation of erythrocytes.

S29434, a Quinone Reductase 2 Inhibitor: Main Biochemical and Cellular Characterization.

Quinone reductase 2 (QR2, E.C. 1.10.5.1) is an enzyme with a feature that has attracted attention for several decades: in standard conditions, instead of recognizing NAD(P)H as an electron donor, it recognizes putative metabolites of NADH, such as N-methyl- and N-ribosyl-dihydronicotinamide. QR2 has been particularly associated with reactive oxygen species and memory, strongly suggesting a link among QR2 (as a possible key element in pro-oxidation), autophagy, and neurodegeneration. In molecular and cellular pharmacology, understanding physiopathological associations can be difficult because of a lack of specific and powerful tools. Here, we present a thorough description of the potent, nanomolar inhibitor [2-(2-methoxy-5H-1,4b,9-triaza(indeno[2,1-a]inden-10-yl)ethyl]-2-furamide (S29434 or NMDPEF; IC50 = 5-16 nM) of QR2 at different organizational levels. We provide full detailed syntheses, describe its cocrystallization with and behavior at QR2 on a millisecond timeline, show that it penetrates cell membranes and inhibits QR2-mediated reactive oxygen species (ROS) production within the 100 nM range, and describe its actions in several in vivo models and lack of actions in various ROS-producing systems. The inhibitor is fairly stable in vivo, penetrates cells, specifically inhibits QR2, and shows activities that suggest a key role for this enzyme in different pathologic conditions, including neurodegenerative diseases.

Terminalia albida treatment improves survival in experimental cerebral malaria through reactive oxygen species scavenging and anti-inflammatory properties.

BACKGROUND: The development of Plasmodium resistance to the last effective anti-malarial drugs necessitates the urgent development of new anti-malarial therapeutic strategies. To this end, plants are an important source of new molecules. The objective of this study was to evaluate the anti-malarial effects of Terminalia albida, a plant used in Guinean traditional medicine, as well as its anti-inflammatory and antioxidant properties, which may be useful in treating cases of severe malaria. METHODS: In vitro antiplasmodial activity was evaluated on a chloroquine-resistant strain of Plasmodium falciparum (K-1). In vivo efficacy of the plant extract was measured in the experimental cerebral malaria model based on Plasmodium berghei (strain ANKA) infection. Mice brains were harvested on Day 7-8 post-infection, and T cells recruitment to the brain, expression levels of pro- and anti-inflammatory markers were measured by flow cytometry, RT-qPCR and ELISA. Non-malarial in vitro models of inflammation and oxidative response were used to confirm Terminalia albida effects. Constituents of Terminalia albida extract were characterized by ultra-high performance liquid chromatography coupled with high resolution mass spectrometry. Top ranked compounds were putatively identified using plant databases and in silico fragmentation patterns. RESULTS: In vitro antiplasmodial activity of Terminalia albida was confirmed with an IC50 of 1.5 µg/mL. In vivo, Terminalia albida treatment greatly increased survival rates in P. berghei-infected mice. Treated mice were all alive until Day 12, and the survival rate was 50% on Day 20. Terminalia albida treatment also significantly decreased parasitaemia by 100% on Day 4 and 89% on Day 7 post-infection. In vivo anti-malarial activity was related to anti-inflammatory properties, as Terminalia albida treatment decreased T lymphocyte recruitment and expression of pro-inflammatory markers in brains of treated mice. These properties were confirmed in vitro in the non-malarial model. In vitro, Terminalia albida also demonstrated a remarkable dose-dependent neutralization activity of reactive oxygen species. Twelve compounds were putatively identified in Terminalia albida stem bark. Among them, several molecules already identified may be responsible for the different biological activities observed, especially tannins and triterpenoids. CONCLUSION: The traditional use of Terminalia albida in the treatment of malaria was validated through the combination of in vitro and in vivo studies.

Antileishmanial Compounds Isolated from Psidium Guajava L. Using a Metabolomic Approach.

With an estimated annual incidence of one million cases, leishmaniasis is one of the top five vector-borne diseases. Currently available medical treatments involve side effects, including toxicity, non-specific targeting, and resistance development. Thus, new antileishmanial chemical entities are of the utmost interest to fight against this disease. The aim of this study was to obtain potential antileishmanial natural products from Psidium guajava leaves using a metabolomic workflow. Several crude extracts from P. guajava leaves harvested from different locations in the Lao People's Democratic Republic (Lao PDR) were profiled by liquid chromatography coupled to high-resolution mass spectrometry, and subsequently evaluated for their antileishmanial activities. The putative active compounds were highlighted by multivariate correlation analysis between the antileishmanial response and chromatographic profiles of P. guajava mixtures. The results showed that the pooled apolar fractions from P. guajava were the most active (IC50 = 1.96 ± 0.47 µg/mL). Multivariate data analysis of the apolar fractions highlighted a family of triterpenoid compounds, including jacoumaric acid (IC50 = 1.318 ± 0.59 µg/mL) and corosolic acid (IC50 = 1.01 ± 0.06 µg/mL). Our approach allowed the identification of antileishmanial compounds from the crude extracts in only a small number of steps and can be easily adapted for use in the discovery workflows of several other natural products.

Antimalarial Properties of Dunnione Derivatives as NQO2 Substrates.

Dunnione, a natural product isolated from the leaves of Streptocarpus dunnii (Gesneriaceae), acts as a substrate for quinone-reductases that may be associated with its antimalarial properties. Following our exploration of reactive oxygen species-producing compounds such as indolones, as possible new approaches for the research of new ways to treat this parasitosis, we explored derivatives of this natural product and their possible antiplasmodial and antimalarial properties, in vitro and in vivo, respectively. Apart from one compound, all the products tested had weak to moderate antiplasmodial activities, the best IC50 value being equal to 0.58 µM. In vivo activities in the murine model were moderate (at a dose of 50 mg/kg/mice, five times higher than the dose of chloroquine). These results encourage further pharmacomodulation steps to improve the targeting of the parasitized red blood cells and antimalarial activities.

Adaptation of a microbead assay for the easy evaluation of traditional anti-sickling medicines: application to DREPANOSTAT and FACA.

CONTEXT: Sickle cell disease is a common inherited blood disorder affecting millions of people worldwide. Due to lack of progress in drug discovery for a suitable treatment, sufferers often turn to traditional medicines that take advantage of the plant extracts activity used by traditional healers. OBJECTIVE: This study optimizes an anti-sickling screening test to identify preparations capable of reverting sickle cells back to the morphology of normal red blood cells. We focused on the miniaturization and practicability of the assay, so that it can be adapted to the laboratory conditions commonly found in less developed countries. MATERIALS AND METHODS: We tested two traditional anti-sickling herbal medicines, FACA® and DREPANOSTAT®, composed of Zanthoxylum zanthoxyloides (Lam.) Zepern. & Timler (Rutaceae) and Calotropis procera (Aiton) Dryand. (Apocynaceae) at screening concentrations of hydroethanol extracts from 0.2 to 1 mg/mL. Potential bioactive molecules present in the extracts were profiled using Ultra High Performance Liquid Chromatography coupled with High Resolution Mass Spectrometry (UHPLC-HRMS/MS) method, identified through HRMS, MS/MS spectra and in silico fragmentation tools. RESULTS: Hydroethanol extracts of FACA® and DREPANOSTAT® showed low anti-sickling activity, inhibiting less than 10% of the sickling process. The UHPLC-HRMS/MS profiles identified 28 compounds (18 in FACA® and 15 in DREPANOSTAT®, including common compounds) among which l-phenylalanine is already described as potential anti-sickling agent. When used as positive control, 7 mg/mL phenylalanine reduced the sickled RBC to 52%. DISCUSSION AND CONCLUSIONS: This assay has been optimized for the easy screening of plant extracts or extracted compounds from bioassay guided fractionation, valuable to laboratories from less developed countries.

Role of Quinone Reductase 2 in the Antimalarial Properties of Indolone-Type Derivatives.

Indolone-N-oxides have antiplasmodial properties against Plasmodium falciparum at the erythrocytic stage, with IC50 values in the nanomolar range. The mechanism of action of indolone derivatives involves the production of free radicals, which follows their bioreduction by an unknown mechanism. In this study, we hypothesized that human quinone reductase 2 (hQR2), known to act as a flavin redox switch upon binding to the broadly used antimalarial chloroquine, could be involved in the activity of the redox-active indolone derivatives. Therefore, we investigated the role of hQR2 in the reduction of indolone derivatives. We analyzed the interaction between hQR2 and several indolone-type derivatives by examining enzymatic kinetics, the substrate/protein complex structure with X-ray diffraction analysis, and the production of free radicals with electron paramagnetic resonance. The reduction of each compound in cells overexpressing hQR2 was compared to its reduction in naïve cells. This process could be inhibited by the specific hQR2 inhibitor, S29434. These results confirmed that the anti-malarial activity of indolone-type derivatives was linked to their ability to serve as hQR2 substrates and not as hQR2 inhibitors as reported for chloroquine, leading to the possibility that substrate of hQR2 could be considered as a new avenue for the design of new antimalarial compounds.

Free Superoxide is an Intermediate in the Production of H2O2 by Copper(I)-Aß Peptide and O2.

Oxidative stress is considered as an important factor and an early event in the etiology of Alzheimer's disease (AD). Cu bound to the peptide amyloid-ß (Aß) is found in AD brains, and Cu-Aß could contribute to this oxidative stress, as it is able to produce in vitro H2O2 and HO˙ in the presence of oxygen and biological reducing agents such as ascorbate. The mechanism of Cu-Aß-catalyzed H2O2 production is however not known, although it was proposed that H2O2 is directly formed from O2 via a 2-electron process. Here, we implement an electrochemical setup and use the specificity of superoxide dismutase-1 (SOD1) to show, for the first time, that H2O2 production by Cu-Aß in the presence of ascorbate occurs mainly via a free O2˙(-) intermediate. This finding radically changes the view on the catalytic mechanism of H2O2 production by Cu-Aß, and opens the possibility that Cu-Aß-catalyzed O2˙(-) contributes to oxidative stress in AD, and hence may be of interest.

Melatonin facts: Melatonin lacks immuno-inflammation boosting capacities at the molecular and cellular levels.

Among the properties melatonin is claimed to possess, are the immuno-inflammation inductive capacities that would be responsible of some of the paramount of activities melatonin is reported to have in most of the human pathological conditions. In the present paper, we measured the effect of melatonin on established cellular models of immuno-inflammation, and found none. The discrepancies are discussed, especially because those properties are reported at pharmacological concentration (1 µM and beyond) at which the melatonin receptors are desensitized by internalization, leading to putative non-receptor-dependent mechanism of action.

Autophagy and neuroprotection in astrocytes exposed to 6-hydroxydopamine is negatively regulated by NQO2: relevance to Parkinson's disease.

Dopaminergic degeneration is a central feature of Parkinson's disease (PD), but glial dysfunction may accelerate or trigger neuronal death. In fact, astrocytes play a key role in the maintenance of the blood-brain barrier and detoxification. 6-hydroxydopamine (6OHDA) is used to induce PD in rodent models due to its specific toxicity to dopaminergic neurons, but its effect on astrocytes has been poorly investigated. Here, we show that 6OHDA dose-dependently impairs autophagy in human U373 cells and primary murine astrocytes in the absence of cell death. LC3II downregulation was observed 6 to 48 h after treatment. Interestingly, 6OHDA enhanced NRH:quinone oxidoreductase 2 (NQO2) expression and activity in U373 cells, even if 6OHDA turned out not to be its substrate. Autophagic flux was restored by inhibition of NQO2 with S29434, which correlated with a partial reduction in oxidative stress in response to 6OHDA in human and murine astrocytes. NQO2 inhibition also increased the neuroprotective capability of U373 cells, since S29434 protected dopaminergic SHSY5Y cells from 6OHDA-induced cell death when cocultured with astrocytes. The toxic effects of 6OHDA on autophagy were attenuated by silencing NQO2 in human cells and primary astrocytes from NQO2-/- mice. Finally, the analysis of Gene Expression Omnibus datasets showed elevated NQO2 gene expression in the blood cells of early-stage PD patients. These data support a toxifying function of NQO2 in dopaminergic degeneration via negative regulation of autophagy and neuroprotection in astrocytes, suggesting a potential pharmacological target in PD.

Resistance to artemisinin in falciparum malaria parasites: A redox-mediated phenomenon.

Malaria remains a major public health disease due to its high yearly mortality and morbidity. Resistance to the gold standard drug, artemisinin, is worrisome and needs better understanding in order to be overcome. In this work, we sought to study whether redox processes are involved in artemisinin resistance. As artemisinin is known to act among others via production of reactive species, we first compared the production of reactive oxygen species and concomitant protein oxidation in artemisinin-sensitive and artemisinin-resistant parasites when treated with artemisinin. The results undoubtedly demonstrated, using different original methods, that the level of ROS, including superoxide production, and oxidized protein were lower in the resistant strain. Interestingly, the major in-between strain difference was reported at the earlier ring stages, which are the forms able to enter in a quiescence state according to the ART resistance phenomenon. Moreover, we demonstrated a better homeostasis regulation in relation with higher expression of antioxidants in the artemisinin-resistant parasites than their sensitive counterparts after artemisinin exposure, notably, superoxide dismutase and the glutathione (GSH) system. These findings enrich the body of knowledges about the multifaceted mechanism of artemisinin resistance and will help in the design and development of newer antimalarials strategies active against resistant parasites.

In Vitro and In Silico Antimalarial Evaluation of FM-AZ, a New Artemisinin Derivative.

Artemisinin-based Combination Therapies (ACTs) are currently the frontline treatment against Plasmodium falciparum malaria, but parasite resistance to artemisinin (ART) and its derivatives, core components of ACTs, is spreading in the Mekong countries. In this study, we report the synthesis of several novel artemisinin derivatives and evaluate their in vitro and in silico capacity to counteract Plasmodium falciparum artemisinin resistance. Furthermore, recognizing that the malaria parasite devotes considerable resources to minimizing the oxidative stress that it creates during its rapid consumption of hemoglobin and the release of heme, we sought to explore whether further augmentation of this oxidative toxicity might constitute an important addition to artemisinins. The present report demonstrates, in vitro, that FM-AZ, a newly synthesized artemisinin derivative, has a lower IC50 than artemisinin in P. falciparum and a rapid action in killing the parasites. The docking studies for important parasite protein targets, PfATP6 and PfHDP, complemented the in vitro results, explaining the superior IC50 values of FM-AZ in comparison with ART obtained for the ART-resistant strain. However, cross-resistance between FM-AZ and artemisinins was evidenced in vitro.

Reactive Oxygen Species as the Brainbox in Malaria Treatment.

Several measures are in place to combat the worldwide spread of malaria, especially in regions of high endemicity. In part, most common antimalarials, such as quinolines and artemisinin and its derivatives, deploy an ROS-mediated approach to kill malaria parasites. Although some antimalarials may share similar targets and mechanisms of action, varying levels of reactive oxygen species (ROS) generation may account for their varying pharmacological activities. Regardless of the numerous approaches employed currently and in development to treat malaria, concerningly, there has been increasing development of resistance by Plasmodium falciparum, which can be connected to the ability of the parasites to manage the oxidative stress from ROS produced under steady or treatment states. ROS generation has remained the mainstay in enforcing the antiparasitic activity of most conventional antimalarials. However, a combination of conventional drugs with ROS-generating ability and newer drugs that exploit vital metabolic pathways, such antioxidant machinery, could be the way forward in effective malaria control.

Superoxide: A major role in the mechanism of action of essential antimalarial drugs.

Understanding the mode of action of antimalarials is central to optimizing their use and the discovery of new therapeutics. Currently used antimalarials belong to a limited series of chemical structures and their mechanisms of action are coutinuously debated. Whereas the involvement of reactive species that in turn kill the parasites sensitive to oxidative stress, is accepted for artemisinins, little is known about the generation of such species in the case of quinolines or hydroxynaphtoquinone. Moreover, the nature of the reactive species involved has never been characterized in Plasmodium-infected erythrocytes. The aim of this work was to determine and elucidate the production of the primary radical, superoxide in Plasmodium-infected erythrocytes treated with artemisinin, dihydroartemisinin, chloroquine and atovaquone, as representatives of three major classes of antimalarials. The intracellular generation of superoxide was quantified by liquid chromatography coupled to mass spectrometry (LC-MS). We demonstrated that artemisinins, atovaquone and to a lesser extent chloroquine, generate significant levels of superoxide radicals in Plasmodium falciparum sensitive strains. More so, the production of superoxide was lowered in chloroquine-resistant strain of Plasmodium treated with chloroquine. These results consolidate the knowledge about the mechanism of action of these different antimalarials and should be taken into consideration in the design of future drugs to fight drug-resistant parasites.

Association of NQO2 With UDP-Glucuronosyltransferases Reduces Menadione Toxicity in Neuroblastoma Cells.

The balance between detoxification and toxicity is linked to enzymes of the drug metabolism Phase I (cytochrome P450 or oxidoreductases) and phase II conjugating enzymes (such as the UGTs). After the reduction of quinones, the product of the reaction, the quinols-if not conjugated-re-oxidizes spontaneously to form the substrate quinone with the concomitant production of the toxic reactive oxygen species (ROS). Herein, we documented the modulation of the toxicity of the quinone menadione on a genetically modified neuroblastoma model cell line that expresses both the quinone oxidoreductase 2 (NQO2, E.C. 1.10.5.1) alone or together with the conjugation enzyme UDP-glucuronosyltransferase (UGT1A6, E.C. 2.4.1.17), one of the two UGT isoenzymes capable to conjugate menadione. As previously shown, NQO2 enzymatic activity is concomitant to massive ROS production, as previously shown. The quantification of ROS produced by the menadione metabolism was probed by electron-paramagnetic resonance (EPR) on cell homogenates, while the production of superoxide was measured by liquid chromatography coupled to mass spectrometry (LC-MS) on intact cells. In addition, the dysregulation of the redox homeostasis upon the cell exposure to menadione was studied by fluorescence measurements. Both EPR and LCMS studies confirmed a significant increase in the ROS production in the NQO2 overexpressing cells due to the fast reduction of quinone into quinol that can re-oxidize to form superoxide radicals. However, the effect of NQO2 inhibition was drastically different between cells overexpressing only NQO2 vs. both NQO2 and UGT. Whereas NQO2 inhibition decreases the amount of superoxide in the first case by decreasing the amount of quinol formed, it increased the toxicity of menadione in the cells co-expressing both enzymes. Moreover, for the cells co-expressing QR2 and UGT the homeostasis dysregulation was lower in presence of menadione than for the its counterpart expressing only QR2. Those results confirmed that the cooperation of the two enzymes plays a fundamental role during the cells' detoxification process. The fluorescence measurements of the variation of redox homeostasis of each cell line and the detection of a glucuronide form of menadiol in the cells co-expressing NQO2 and UGT1A6 enzymes further confirmed our findings.

Effect of Silica Based Nanoparticles against Plasmodium falciparum and Leishmania infantum parasites.

Malaria and Leishmaniasis are two major parasitic diseases, endemic in large areas of tropical countries with high morbidity and mortality across the world. Nanoparticles in small sizes are specifically considered in medicine due to their ability to enter the cells, control the distribution of the administered drug and carry the drug specifically to the place of action. The present study aims to introduce the application of silica nanoparticles as new promising nanotools in malaria and leishmaniasis treatment. Ion doped silica nanomaterials revealed antileishmanial activities indicating the positive role of calcium, magnesium and copper to the surface of the particles against Leishmania parasites. Artemisinin-loaded nanoparticles presented the most promising antiparasitic properties with a sustained release able to overcome the parasite invasion. The sustainable release of artemisinin guarantee both the maintenance of its potential efficacy and also introduce an administration of drug to avoid subsequent drug resistance.

Effect of Artemisinin-Loaded Mesoporous Cerium-Doped Calcium Silicate Nanopowder on Cell Proliferation of Human Periodontal Ligament Fibroblasts.

Ion doping has rendered mesoporous structures important materials in the field of tissue engineering, as apart from drug carriers, they can additionally serve as regenerative materials. The purpose of the present study was the synthesis, characterization and evaluation of the effect of artemisinin (ART)-loaded cerium-doped mesoporous calcium silicate nanopowders (NPs) on the hemocompatibility and cell proliferation of human periodontal ligament fibroblasts (hPDLFs). Mesoporous NPs were synthesized in a basic environment via a surfactant assisted cooperative self-assembly process and were characterized using Scanning Electron Microscopy (SEM), X-ray Fluorescence Spectroscopy (XRF), Fourier Transform Infrared Spectroscopy (FT-IR), X-ray Diffraction Analysis (XRD) and N2 Porosimetry. The loading capacity of NPs was evaluated using Ultrahigh Performance Liquid Chromatography/High resolution Mass Spectrometry (UHPLC/HRMS). Their biocompatibility was evaluated with the MTT assay, and the analysis of reactive oxygen species was performed using the cell-permeable ROS-sensitive probe 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA). The synthesized NPs presented a mesoporous structure with a surface area ranging from 1312 m2/g for undoped silica to 495 m2/g for the Ce-doped NPs, excellent bioactivity after a 1-day immersion in c-SBF, hemocompatibility and a high loading capacity (around 80%). They presented ROS scavenging properties, and both the unloaded and ART-loaded NPs significantly promoted cell proliferation even at high concentrations of NPs (125 µg/mL). The ART-loaded Ce-doped NPs with the highest amount of cerium slightly restricted cell proliferation after 7 days of culture, but the difference was not significant compared with the control untreated cells.

An LC-MS Assay to Measure Superoxide Radicals and Hydrogen Peroxide in the Blood System.

Red blood cells are constantly exposed to reactive species under physiological or pathological conditions or during administration of xenobiotics. Regardless of the source, its accurate quantification is paramount in the area of theragnostics, which had been elusive up until now. Even if there are a lot of approaches to evaluate the oxidative stress, very sensitive methods are missing for the blood system. We therefore sought to apply a highly sensitive approach, by liquid chromatography coupled to mass spectrometry (UPLC-MS), for the quantification of reactive species such as superoxide radical and hydrogen peroxide using dihydroethidium (DHE) and coumarin boronic acid (CBA) probes respectively through the detection of 2-hydroxyethidium (2OH-E+) and 7-hydroxycoumarin (COH). The use of the high-resolution mass spectrometry associated to UPLC ensured a selective detection of superoxide and hydrogen peroxide in the blood system under diverse conditions such as oxidized red blood cells (RBCs), untreated and treated parasitized RBCs. Moreover, this technique allowed the determination of reactive species in human plasma. This protocol provides a huge opportunity for in-depth study of several pathological conditions vis-a-vis their treatment in modern medicine.

Syk Kinase Inhibitors Synergize with Artemisinins by Enhancing Oxidative Stress in Plasmodium falciparum-Parasitized Erythrocytes.

Although artemisinin-based combination therapies (ACTs) treat Plasmodium falciparum malaria effectively throughout most of the world, the recent expansion of ACT-resistant strains in some countries of the Greater Mekong Subregion (GMS) further increased the interest in improving the effectiveness of treatment and counteracting resistance. Recognizing that (1) partially denatured hemoglobin containing reactive iron (hemichromes) is generated in parasitized red blood cells (pRBC) by oxidative stress, (2) redox-active hemichromes have the potential to enhance oxidative stress triggered by the parasite and the activation of artemisinin to its pharmaceutically active form, and (3) Syk kinase inhibitors block the release of membrane microparticles containing hemichromes, we hypothesized that increasing hemichrome content in parasitized erythrocytes through the inhibition of Syk kinase might trigger a virtuous cycle involving the activation of artemisinin, the enhancement of oxidative stress elicited by activated artemisinin, and a further increase in hemichrome production. We demonstrate here that artemisinin indeed augments oxidative stress within parasitized RBCs and that Syk kinase inhibitors further increase iron-dependent oxidative stress, synergizing with artemisinin in killing the parasite. We then demonstrate that Syk kinase inhibitors achieve this oxidative enhancement by preventing parasite-induced release of erythrocyte-derived microparticles containing redox-active hemichromes. We also observe that Syk kinase inhibitors do not promote oxidative toxicity to healthy RBCs as they do not produce appreciable amounts of hemichromes. Since some Syk kinase inhibitors can be taken daily with minimal side effects, we propose that Syk kinase inhibitors could evidently contribute to the potentiation of ACTs.

Effect of Sintering Temperature of Bioactive Glass Nanoceramics on the Hemolytic Activity and Oxidative Stress Biomarkers in Erythrocytes.

INTRODUCTION: The nature of the surface is critical in determining the biological activity of silica powders. A novel correlation between toxicity and surface properties of bioactive glass ceramics (BGCs) synthesized via the sol-gel method was attempted in this study. METHODS: The behavior of BGCs after their attachment to the surface of red blood cells (RBCs) was evaluated and their toxic effects were determined based on hemolysis, membrane injury via anti-phosphotyrosine immunoblot of Band 3, lipid peroxidation, potential to generate reactive oxygen species, and antioxidant enzyme production. In particular, three BGCs were synthesized and treated at three sintering temperatures (T1 = 835 °C, T2 = 1000 °C and T3 = 1100 °C) to investigate possible relation between surface charge or structure and hemolytic potential. RESULTS: Their toxicity based on hemolysis was dose dependent, while BGC-T2 had the best hemocompatibility in compare with the other BGCs.No BGCs in dosages lower than 0.125 mg/mL could damage erythrocytes. On the other hand, all BGCs promoted the production of reactive oxygen species in certain concentrations, with the BGC-T2 producing the lowest ROS and increasing the glutathione levels in RBCs protecting their damage. CONCLUSIONS: The results suggest that various factors such as size, a probable different proportion of surface silanols, a balanced mechanism between calcium and magnesium cellular uptake or the different crystalline nature may have contributed to this finding; however, future research is needed to clarify the underlying mechanisms.