Introduction

The role of reactive oxygen species (ROS) generated by polymorphonuclear leucocytes (PMNs) in the host response against malaria was investigated. Non-activated human PMNs were added to cultures of P. falciparum in microtitre cells. Parasite viability was evaluated by the incorporation of radioactive hypoxanthine. Using PMN/RBC = 1/150 (starting parasitemia was 1+) the incorporation on the second day in culture was only 61+ of the control cultures. An effect could be observed already after two hours of incubation (30+ reduction at a 1/50 PMN/RBC ratio). A direct contact between the effector and target cells was obligatory for the expression of the damage.

Parasites within G6PD-deficient erythrocytes were more sensitive to the PMNs than normal parasitized erythrocytes. This difference could be attributed to the production of reactive oxygen intermediates in the experimental system, since G6PD-deficient erythrocytes are generally more sensitive to oxidant stress.

Salicylic acid was used as a scavenger and reporter molecule for hydroxyl radical fluxes. It is converted to the corresponding dihydroxybenzoic acid derivatives, which could be detected by HPLC. Uninfected NRBC or parasitized erythrocytes containing young ring forms could trigger the PMNs to produce much less ROS than the mature forms of the parasites. Other factors associated with PMNs may inactivate the parasites, such as phagocytosis, lysosomal enzymes or degradation toxic products of the PMNs. However our results indicate that increased oxidative stress induced by PMNs interfere with the growth of P. falciparum and could play a role in human evolution of abnormal erythrocytes.     

A rapid, simple and sensitive flow cytometric system for detection of Plasmodium falciparum.

We have established a rapid, simple and sensitive flow cytometric system for the detection of Plasmodium falciparum that involves lysing erythrocytes and staining parasites at the same time using a newly developed hemolysing and staining solution containing dodecyl methyl ammonium chloride and acridine orange. In this system, freed parasites of P. falciparum could be plotted separately from erythrocyte ghosts, white blood cells and platelets on the two-dimensional scattergram of forward-angle light scatter and green fluorescence by flow cytometry with an argon laser. It took only 2–3 min per sample to obtain the scattergram and analyze the data, including the time of sample preparation for flow cytometric analysis. Sample preparation with this method does not require any difficult handling procedures. The threshold of parasite detection was almost equal to that of microscopic examination for cultured P. falciparum. The results of drug-susceptibility assays using this system were also almost identical to those obtained using microscopic examination. In this system, parasites at different erythrocytic stages could be easily distinguished. This system must prove useful and practical for basic laboratory studies of P. falciparum including those requiring the differential measurement of parasites at specific erythrocytic stages.     

The Effect of Free Radicals Induced by Paraquat and Copper on the In Vitro Development of Plasmodium falciparum

The role of transition metals in paraquat toxicity was studied in cultures of Plasmodium falciparum. We showed that addition of copper led to an enhancement of the plasmodium killing, whereas addition of chelating agents. such as desferrioxamine and diethylenetriamine pentaacetic acid markedly reduced the toxic effects. Parsitized G6PD deficient erythrocytes were more sensitive than parasitized normal eryth-rocytes to copper and to the combination of copper and paraquat.     

Gametocytocidal activity of pyronaridine and DNA topoisomerase II inhibitors against multidrug-resistant Plasmodium falciparum in vitro

Gametocytocidal activities of pyronaridine and DNA topoisomerase II inhibitors against two isolates of multidrug-resistant Plasmodium falciparum, KT1 and KT3 were determined. After sorbitol treatment, pure gametocyte cultures of Plasmodium falciparum containing mostly young gametocytes (stage II and III) obtained on day 11 were exposed to the drugs for 48 h. The effect of the drugs on gametocyte development was assessed by counting gametocytes on day 15 of culture. Pyronaridine was the most effective gametocytocidal drug against P. falciparum isolates KT1 and KT3 with 50% inhibitory concentration of 6 and 20 nM, respectively. Moreover, the 50% inhibitory concentration of pyronaridine was lower than that of primaquine which is the only drug used to treat malaria patients harboring gametocytes. Prokaryotic (norfloxacin) and eukaryotic (amsacrine and etoposide) DNA topoisomerase II inhibitors were only effective against asexual but not sexual stages of the malaria parasites. Pyronaridine has both schizontocidal and gametocytocidal activities against the human malaria parasite, P. falciparum.     

Monoquaternary ammonium derivatives inhibit growth of protozoan parasites

The phospholipid metabolism of Plasmodium falciparum-infected erythrocytes has been shown to be an effective pharmacological target for novel chemotherapy. Thirty-seven monoquaternary ammonium derivatives analogous to choline were screened for their potential antiprotozoal activity against P. falciparum and Leishmania braziliensis. Twenty-three compounds inhibited chloroquine resistant and sensitive P. falciparum strains with inhibitory concentrations ranging from 0.001 μM to 47 μM. Among the inhibitors were six compounds with nanomolar activity containing at least one ethyl group in the polar head and a hydrophobic alkyl chain with 10 to 14 methylene groups. Four compounds also exhibited in vitro antileishmanial properties in the micromolar range.     

Functional Activity of Blood Polymorphonuclear Leukocytes as an Oxidative Stress Biomarker in Human Subjects

In the present work, we studied the role of polymorphonuclear leukocytes (PMN) in aged individuals and coronary heart disease (CHD)-bearing patients, two physiopathological processes associated with overproduction of reactive oxygen species (ROS). The effects of antioxidant supplementation on the functional activity of PMN from CHD patients were also determined. The function of PMNs was evaluated by measuring of phagocytosis, killing activity, and ROS production. Luminol amplified chemiluminescence (CL) was used to estimate ROS production by stimulated PMNs. Total cholesterol and the LDL-cholesterol fraction from CHD patients were found to be higher than those recommended, returning to normal levels after antioxidant therapy. PMN CL of CHD patients was found to be higher than the associated control groups. Antioxidant therapy administrated to CHD patients lead to an increase in the killing activity accompanied by a decrease in PMN CL of these subjects. The study also showed that killing activity of PMN from human subjects over 60 years was significantly lower than the activity measured in younger subjects. PMN CL produced after stimulation was found to be positively correlated with the increasing age of human subjects (r = .946, p < .01).     

Refractoriness of erythrocytes infected with Plasmodium falciparum gametocytes to lysis by sorbitol

Unlike erythrocytes infected with mature asexual parasites of Plasmodium falciparum, those infected with gametoeytes are not lysed by 5% sorbitol solutions. This observation was used to devise a method for producing synchronized cultures of gametocytes, free of asexual stage parasites. The refractoriness to sorbitol suggests that the major anion transport pathway, which appears in the membrane of erythrocytes infected with asexual stage parasites, is not present in cells infected with gametocytes.     

Culture of Plasmodium falciparum: the role of pH, glucose, and lactate

Yields of P. falciparum in intraerythrocytic in vitro cultures were maximized when extracellular pH was maintained between 7.2 and 7.45, and extracellular lactate was kept below 12 mM. Host erythrocytes metabolized 4.6 +/- 1.5 microM glucose/10(9) RBC/24 hr and produced 7.9 +/- 1.8 microM lactate/10(9) RBC/24 hr. Asynchronous parasite cultures used 122 +/- 34 microM glucose/10(9) parasitized RBC/24 hr and produced 143 +/- 47 microM lactate/10(9) parasitized RBC/24 hr. Synchronous cultures that were 80 to 100% ring forms after 24 hr in culture exhibited significantly lower glycolysis per 10(9) parasitized RBC than cultures that were 0 to 25% ring forms after 24 hr. The percent of glucose utilization accounted for by lactate production by parasites was significantly less than that of uninfected erythrocytes. These optimum ranges and metabolic rates can be used in the development of parasite culture techniques.     

Antimalarial properties of soy-bean fat emulsions

Intralipid^® and Ivelip^® are commercial preparations of soy-bean lipid extracts used for intravenous supplementation of lipids in various clinical conditions. They were found to inhibit the growth of Plasmodium falciparum in culture with an IC₅₀ of 8.07 ± 2.13 and 13.32 ± 2.05 mg.ml⁻¹, respectively. Intralipid^® rapidly and efficiently inhibited nucleic acid synthesis in cultured P. falciparum, exhibiting full inhibitory activity in less than 2 h. Ivelip^® injected intraperitoneally, was found by the 4-day suppressive test to be active in vivo against P. vinckei petteri within the normal recommended regimen for dietary lipid supply (0.5–4 g.kg⁻¹), but it was impossible to obtain a radical cure even with very high doses (6.4 g.kg⁻¹). Ivelip^® was less effective against P. berghei and P. yoelii nigeriensis. As Ivelip^® showed no interference with the antimalarial activity of chloroquine, it could be considered for use in the treatment of severe human malaria in association with 4-aminoquinolines to expedite the clearance of parasites.     

Polymorphonuclear leukocytes promote neurotoxicity through release of matrix metalloproteinases, reactive oxygen species, and TNF-alpha

As the first immune cells to infiltrate the nervous system after traumatic PNS and CNS injury, neutrophils (polymorphonuclear leukocytes, PMNs) may promote injury by releasing toxic soluble factors that may affect neuronal survival. Direct neurotoxicity of matrix metalloproteinases (MMPs), reactive oxygen species (ROS), and cytokines released by PMNs was investigated by culturing dorsal root ganglion (DRG) cells with PMN-conditioned media containing MMP inhibitor (GM6001), ROS scavengers, or tumor necrosis factor alphaR (TNF-alphaR) neutralizing antibody. Although DRGs exposed to PMN-conditioned media had 53% fewer surviving neurons than controls, neuronal cell loss was prevented by GM6001 (20 micromol/L), catalase (1000 U/mL), or TNF-alphaR neutralizing antibody (1.5 microg/mL), elevating survival to 77%, 94%, and 95%, respectively. In accordance with protection by GM6001, conditioned media collected from MMP-9 null PMNs was less neurotoxic than that collected from wild-type PMNs. Additionally, MMP inhibition reduced PMN-derived ROS; removal of ROS reduced PMN-derived MMP-9 activity; and TNF-alpha inhibition reduced both PMN-derived MMP-9 activity and ROS in PMN cultures. Our data provide the first direct evidence that PMN-driven neurotoxicity is dependent on MMPs, ROS, and TNF-alpha, and that these factors may regulate PMN release of these soluble factors or interact with one another to mediate PMN-driven neurotoxicity.     

Oxidative Stress as a Defense Mechanism Against Parasitic Infections

Many parasites - including the causative agents of malaria, Chagas' disease and schistosomiasis - are more susceptible to reactive oxygen species (ROS) than their hosts are. This is manifested by one or more of the following criteria: 1. Susceptibility of the parasite to ROS in vitro; 2. macrophage-based defense mechanisms against the parasite in vivo; 3. successful therapy using agents which lead to oxidative stress; 4. selection advantage (with respect to parasite infections) of human populations whose antioxidant capacity is impaired by a gene defect or by strong oxidants in their staple food.

Our laboratory is involved in developing inhibitors against antioxidant enzymes thus mimicking natural experiments. Since glutathione reductase is a protein of known atomic structure the methods of drug design by receptor fit (DDRF) can be applied for this enzyme. Another promising target enzyme is trypanothione reductase which was found so far only in trypanosomatids, and specifically, not in their hosts. Consequently the trypanothione pathway may be a general target in the design of drugs against diseases caused by trypanosomes and leishmanias.     

Baseline in vitro efficacy of ACT component drugs on Plasmodium falciparum clinical isolates from Mali

In vitro susceptibility to antimalarial drugs of Malian Plasmodium falciparum isolates collected between 2004 and 2006 was studied. Susceptibility to chloroquine and to three artemisinin-based combination therapy (ACT) component drugs was assessed as a first, to our knowledge, in vitro susceptibility study in Mali. Overall 96 Malian isolates (51 from around Bamako and 45 collected from French travellers returning from Mali) were cultivated in a CO₂ incubator. Fifty percent inhibitory concentrations (IC₅₀s) were measured by either hypoxanthine incorporation or Plasmodium lactate dehydrogenase (pLDH) ELISA. Although the two sets of data were generated with different methods, the global IC₅₀ distributions showed parallel trends. A good concordance of resistance phenotype with pfcrt 76T mutant genotype was found within the sets of clinical isolates tested. We confirm a high prevalence of P. falciparum in vitro resistance to chloroquine in Mali (60–69%). While some isolates showed IC₅₀s close to the cut-off for resistance to monodesethylamodiaquine, no decreased susceptibility to dihydroartemisinin or lumefantrine was detected. This study provides baseline data for P. falciparum in vitro susceptibility to ACT component drugs in Mali.     

Resistance of Neisseria gonorrhoeae to non-oxidative killing by adherent human polymorphonuclear leucocytes

Symptomatic infection with Neisseria gonorrhoeae (Gc) is characterized by abundant neutrophil (PMN, polymorphonuclear leucocyte) influx, but PMNs cannot clear initial infection, indicating that Gc possess defences against PMN challenge. In this study, survival of liquid-grown Gc was monitored after synchronous infection of adherent, interleukin 8-treated human PMNs. 40–70% of FA1090 Gc survived 1 h of PMN exposure, after which bacterial numbers increased. Assays with bacterial viability dyes along with soybean lectin to detect extracellular Gc revealed that a subset of both intracellular and extracellular PMN-associated Gc were viable. Gc survival was unaffected in PMNs chemically or genetically deficient for producing reactive oxygen species (ROS). This result held true even for OpaB+ Gc, which stimulate neutrophil ROS production. Catalase- and RecA-deficient Gc, which are more sensitive to ROS in vitro , had no PMN survival defect. recN and ngo1686 mutant Gc also exhibit increased sensitivity to ROS and PMNs, but survival of these mutants was not rescued in ROS-deficient cells. The ngo1686 mutant showed increased sensitivity to extracellular but not intracellular PMN killing. We conclude that Gc are remarkably resistant to PMN killing, killing occurs independently of neutrophil ROS production and Ngo1686 and RecN defend Gc from non-oxidative PMN antimicrobial factors.     

In vitro study of the antioxidant properties of non steroidal anti-inflammatory drugs by chemiluminescence and electron spin resonance (ESR)

Objectives. To determine the antioxidant activities of nonsteroidal anti-inflammatory drugs (NSAIDS), we examined by chemiluminescence (CL) and electron spin resonance (ESR) their scavenging properties towards lipid peroxides, hypochlorous acid and peroxynitrite.

Methods. The antioxidant properties of nimesulide (NIM), 4-hydroxynimesulide (4-HONIM), aceclofenac (ACLO), 4-hydroxyaceclofenac (4-HOA-CLO), diclofenac (DICLO) and indomethacin (INDO) were tested on four different reactive oxygen species (ROS) generating systems: (I) phorbol-myristate acetate (PMA)-activated neutrophils, (II) Fe²⁺/ascorbate-induced lipid peroxidation, (III) HOCl-induced light emission, (IV) the kinetics of ONOO^- decomposition followed by spectrophotometry. ROS production was monitored by luminol-enhanced CL or by ESR using two different spin traps.

Results. At 10 μM, ACLO, NIM, 4-HONIM, 4-HOA-CLO, and DICLO decreased luminol-enhanced CL generated by PMA-activated neutrophils. Inversely, INDO increased the luminol enhanced CL. Interestingly, hydroxylated metabolites were more potent antioxidants than the parent drugs. Furthermore, all drugs tested, excepted ACLO, lowered lipid peroxidation induced by Fe²⁺/ascorbate system. ACLO and DICLO, even at the highest concentration tested (100 μM), did not significantly lower HOCl induced CL, whereas the other drugs were potent scavengers. Finally, all the NSAIDS accelerated decomposition of ONOO^-, suggesting a potential capacity of the molecules to scavenge peroxynitrite.

Conclusion. The NSAIDs possess variable degrees of antioxidant activities, linked to their ability to react with HOCl, lipid peroxides or ONOO^-. These antioxidant activities could offer interesting targeted side-effects in the treatment of joint inflammatory diseases.     

Intracellular parasitism: cell biological adaptations of parasitic protozoa to a life inside cells

Several protozoan parasites evade the host's immune defence because most of their development takes place inside specific host cells. Only a few of these protozoa live within the host cell cytosol. Most parasites are sequestered within membrane-bound compartments, collectively called ‘vacuoles’. Recent advances in the cell biology of intracellular parasites have revealed fundamental differences in the strategies whereby such organisms gain entry into their respective host cells. These differences have important implications for host-parasite interaction and for nutrient acquisition by the parasite. Leishmania spp. take advantage of the phagocytic properties of their host cells and presumably contribute little to the uptake process. In contrast, apicomplexan parasites have developed highly specialised organelles, called micronemes and rhoptries, to actively invade a variety of nucleated cells and, in the case of Plasmodium falciparum, human erythrocytes. Following invasion, parasites use a multitude of strategies to protect themselves from the defence mechanisms of the parasitized cells. In addition, they induce novel pathways within the infected cell that allow a most efficient nutrient acquisition both from the host cell cytoplasm and from the extracellular environment. Parasite-induced changes of host cells are most apparent in erythrocytes infected with Plasmodium spp. Mammalian erythrocytes are deficient in de novo protein and lipid biosynthesis and, consequently, pathways which allow the transport of macromolecules and small solutes are established by metabolic activities of the parasite. Research into the cell biology of intracellular parasitism has identified fascinating phenomena some of which we are beginning to understand at a molecular level. They are fascinating because they allow insights into a very intimate interaction between two eukaryotic cells of entirely different phylogenetic origins.     

Role of reactive oxygen species in cardiac preconditioning: study with photoactivated Rose Bengal in isolated rat hearts

Oxygen radical scavengers have been shown to prevent the development of ischemic preconditioning, suggesting that reactive oxygen species (ROS) might be involved in this phenomenon. In the present study, we have investigated whether direct exposure to ROS produced by photoactivated Rose Bengal (RB) could mimic the protective effects of ischemic preconditioning.

Methods In vitro generation of ROS from photoactivated RB in a physiological buffer was first characterised by ESR spectroscopy in the presence of 2,2,6,6-tetramethyl-1-piperidone (oxoTEMP) or 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). In a second part of the study, isolated rat hearts were exposed for 2.5 min to photoactivated RB. After 5 min washout, hearts underwent 30 min no-flow normothermic ischemia followed by 30 min of reperfusion.

Results and Conclusions The production of singlet oxygen (¹O₂) by photoactivated RB in the perfusion medium was evidenced by the ESR detection of the nitroxyl radical oxoTEMPO. Histidine completely inhibited oxoTEMPO formation. In addition, the use of DMPO has indicated that (i) superoxide anions (O^·-₂) are produced directly and (ii) hydroxyl radicals (HO^·) are formed indirectly from the successive O^·-₂ dismutation and the Fenton reaction. In the perfusion experiments, myocardial post-ischemic recovery was dramatically impaired in hearts previously exposed to the ROS produced by RB photoactivation (¹O₂, O^·-₂, H₂O₂ and HO^·) as well as when ¹O₂ was removed by histidine (50 mM) addition. However, functional recovery was significantly improved when hearts were exposed to photoactivated RB in presence of superoxide dismutase (10⁵ IU/L) and catalase (10⁶ IU/L).

Further studies are now required to determine whether the cardioprotective effects of Rose Bengal in presence of O^·-₂ and H₂O₂ scavengers are due to singlet oxygen or to other species produced by Rose Bengal degradation.     

An inducible release of reactive oxygen radicals in four species of gorgonian corals

The capability for physical injury or heat stress to elicit the production of reactive oxygen species was examined in four species of gorgonian corals. The sea plumes Pseudopterogorgia elisabethae, Pseudopterogorgia americana, the sea rod Eunicea fusca and the azooxanthellate red branching gorgonian Lophogorgia chilensis were physically injured using sonic sound cavitations and heat shocked by incubation in 33°C sea water. The pharmacological probe, diphenylene iodonium chloride (DPI), an inhibitor of NAD(P)H oxidase and peroxidases was used to identify an enzymatic surrogate of the oxidative burst. Both injury and heat stress were capable of inducing the release of reactive oxygen species (ROS) in all gorgonians tested, yet the kinetics and amplitude of ROS release varied among genera. In both the treatments, P. americana demonstrated the largest oxidative burst among the other corals tested.     

Refractoriness of eryptotic red blood cells to Plasmodium falciparum infection: a putative host defense mechanism limiting parasitaemia

Recently, we have described that apoptosis-like process of red blood cells (RBC) - eryptosis - in malaria is not restricted to parasitized cells, occurring also in non-parasitized RBC (nRBC). Besides to pathogenic proprieties, apoptosis also participates in the innate defense trough restriction of intracellular pathogens propagation. In the present study, we investigated the capacity of P. falciparum parasites to infect eryptotic RBC. Schizont parasitized RBC concentrated by magnetic separation were cultured with eryptotic RBC obtained by ionomycin treatment and, then, parasite growth was evaluated in Giemsa-stained thin blood smears. While parasites infected and developed normally in control non-eryptotic RBC, cultures performed with eryptotic RBC had a marked decrease in parasitaemia. It was noteworthy a great number of free merozoites in eryptotic RBC cultures, indicating that these cells were not susceptible to invasion. We suggest that although eryptosis could be involved in malaria pathogenesis, it could also acting protectively by controlling parasite propagation.     

A novel effect of polymorphonuclear leukocytes in the facilitation of angiogenesis

The purpose of this study was to examine whether the adhesion of polymorphonuclear leukocytes (PMNs) to endothelial cells and/or reactive oxygen species (ROS) released from PMNs are responsible for inducing angiogenesis. Angiogenesis was assessed by tube formation using endothelial cells obtained from bovine thoracic aorta (BAECs) grown on a layer of collagen type I. Addition of PMNs to BAECs weakly induced angiogenesis. The angiogenesis induced by PMNs alone was further enhanced by treatment of the PMNs with N-formyl-methionyl-leucyl-phenylalanine (FMLP), a selective activator of PMN. The involvement of PMN adhesion to BAECs via adhesion molecules in angiogenesis was investigated by using monoclonal antibodies against E-selectin and intercellular adhesion molecule-1 (ICAM-1). These antibodies blocked both the PMN adhesion to BAECs and the enhancement of angiogenesis induced by FMLP-treated PMNs. Furthermore, the enhancement of angiogenesis by FMLP-treated PMNs was blocked by catalase, a scavenging enzyme of H2O2, but not by superoxide dismutase (SOD). These results suggest that PMNs induce angiogenesis in vitro, and that the mechanism of stimulation of angiogenesis by PMNs may involve the adherence of PMNs to endothelial cells via E-selectin and ICAM-1, and H2O2, but not superoxide. Thus, activated PMNs in pathological states may not only induce tissue injury, but may also function as regulators of angiogenesis.     

Marginated pool of neutrophils in rabbit lungs

The size and location of the marginated pool of neutrophils (PMNs) in rabbit lungs were evaluated, and the rate of exchange of the PMNs with the circulating pool was determined. 99mTc-labeled erythrocytes (99mTc-RBCs) and 125I-labeled macroaggregated albumin (125I-MAA) were used to determine RBC transit times in the pulmonary circulation. Radiolabeled PMNs were studied on their first passage through the lungs. After 10 min of circulation, the lungs were fixed, gamma counted, and prepared for morphometric and autoradiographic studies; 74 +/- 3% of the PMNs was retained in the lungs on the first passage, and 23 +/- 2% was within the pulmonary marginated pool 10 min later. The regional PMN retention and the rate of exchange between the marginated and circulating PMN pools in the lung were directly related to RBC transit time. The radiolabeled PMNs distributed similarly to the unlabeled cells within the microvasculature and had a similar exchange rate between the marginated and circulating pools (1.4 +/- 0.2%/s using labeled cells and 1.5 +/- 0.5%/s using unlabeled cells). The marginated pool was located primarily within alveolar capillaries and contained two to three times as many PMNs as the total circulating pool.