Killing of Plasmodium falciparum in vitro by nitric oxide derivatives.

We have investigated the in vitro susceptibility of the human malaria parasite Plasmodium falciparum to killing by nitric oxide and related molecules. A saturated solution of nitric oxide did not inhibit parasite growth, but two oxidation products of nitric oxide (nitrite and nitrate ions) were toxic to the parasite in millimolar concentrations. Nitrosothiol derivatives of cysteine and glutathione were found to be about a thousand times more active (50% growth inhibitory concentration, approximately 40 microM) than nitrite.     

L-arginine metabolism and its impact on host immunity against <Emphasis Type="Italic">Leishmania</Emphasis> infection

Leishmaniasis is a vector-borne disease found in many countries worldwide. The causative agent of the disease, Leishmania spp., lives as an obligate intracellular parasite within mammalian hosts. Since tissue macrophages are major target cells for parasite replication, the outcome of infection depends largely on the activation status of these cells. L-arginine is a crucial amino acid required for both nitric oxide (NO)-mediated parasite killing and polyamine-mediated parasite replication. This review highlights the significance of L-arginine as a factor determining the outcomes of Leishmania infection in vitro and its influences on host immune responses in vivo. Various therapeutic approaches targeting L-arginine metabolic pathways during infections with Leishmania are also discussed.     

Glycoinositolphospholipids of Leishmania major inhibit nitric oxide synthesis and reduce leishmanicidal activity in murine macrophages

Murine macrophages express high levels of inducible nitric oxide (NO) synthase and produce large amounts of nitric oxide when activated with interferon-γ and lipopolysaccharide in vitro. Nitric oxide is a mediator of a variety of biological functions including microbicidal activity against the protozoan parasite Leishmania species. Glycoinositolphospholipids (GIPL) are the predominant surface glycolipids in both developmental stages of Leishmania major. We report here that GIPL can inhibit the synthesis of NO in a time- and dose-dependent manner. In contrast, lipophosphoglycan, which is present in the promastigote stage did not inhibit NO synthesis. GIPL-treated macrophages also showed markedly reduced leishmanicidal activity. The majority of the inhibitory activity of GIPL was found within the alkylacylglycerol moiety of the GIPL molecule. These data, therefore, suggest that GIPL may contribute towards the survival of the parasite in the immune hosts.     

Neurocysticercosis, IgG immunoglobulins, and nitric oxide

This study evaluated the role of parasite load and nitric oxide on IgG levels in neurocysticercosis. Total serum IgG, IgG antibodies specific for cysticercus antigens, and nitric oxide were compared between 85 neurocysticercosis patients, 65 with solitary cysts and 20 with multiple cysts, and 13 normal healthy controls. Sixty-six percent of patients were seropositive for cysticercus IgG antibodies. Among seropositive patients, IgG levels did not differ between those infected with multiple or solitary cysts whose serum nitric oxide levels were low (<40 nmol/ml). Among seropositive solitary cyst infected patients, IgG levels were significantly higher in those whose serum nitric oxide was low compared to those with high nitric oxide levels (p < 0.001). IgG levels were significantly higher in patients with multiple compared to single cyst infections among those negative for cysticercus antibodies (p < 0.001). Parasite load and nitric oxide modulated IgG production in neurocysticercosis. IgG levels were not determined by the number of infecting cysts in seropositive patients who did not mount a nitric oxide response. IgG production correlated to parasite load in patients negative for cysticercus antibodies.     

Toll-like receptor 4 contributes to efficient control of infection with the protozoan parasite Leishmania major

The essential role of Toll-like receptors (TLR) in innate immune responses to bacterial pathogens is increasingly recognized, but very little is known about the role of TLRs in host defense against infections with eukaryotic pathogens. For the present study, we investigated whether TLRs contribute to the innate and acquired immune response to infection with the intracellular protozoan parasite Leishmania major. Our results show that TLR4 contributes to the control of parasite growth in both phases of the immune response. We also addressed the mechanism that results in killing or growth of the intracellular parasites. Control of parasite replication correlates with the early induction of inducible nitric oxide synthase in TLR4-competent mice, whereas increased parasite survival in host cells from TLR4-deficient mice correlates with a higher activity of arginase, an enzyme known to promote parasite growth. This is the first study showing that TLR4 contributes to the effective control of Leishmania infection in vivo.     

Cellular mechanisms of nonspecific immunity to intracellular infection: cytokine-induced synthesis of toxic nitrogen oxides from L-arginine by macrophages and hepatocytes

Nitric oxide (NO) produced by cytokine-treated macrophages and hepatocytes plays a vital role in protective host responses to infectious pathogens. NO inhibits iron-sulfur-dependent enzymes involved in cellular respiration, energy production, and reproduction. Synthesis of L-arginine-derived nitrite (NO2-), the oxidative end product of NO, directly correlates with intracellular killing of Leishmania major, an obligate intracellular protozoan parasite of macrophages: the level of NO2- production is a quantitative index for macrophage activation. The competitive inhibitor of NO synthesis, monomethylarginine (NGMMLA), inhibits both parasite killing and NO2- production. For Leishmania, the parasite itself participates in the regulation of this toxic effector mechanism. This participation is mediated by parasite induction of tumor necrosis factor alpha (TNF alpha), an autocrine factor of macrophages: NO synthesis by interferon-gamma (IFN-gamma)-treated cells can be blocked by monoclonal antibodies to TNF alpha. NO production by IFN gamma-treated hepatocytes is of special interest in malaria infections: sporozoite-infected hepatocytes kill the intracellular malaria parasite after treatment with IFN gamma; this killing is inhibited by NGMMLA.     

Induction of nitric oxide synthase in Anopheles stephensi by Plasmodium falciparum: mechanism of signaling and the role of parasite glycosylphosphatidylinositols

Malaria parasite (Plasmodium spp.) infection in the mosquito Anopheles stephensi induces significant expression of A. stephensi nitric oxide synthase (AsNOS) in the midgut epithelium as early as 6 h postinfection and intermittently thereafter. This induction results in the synthesis of inflammatory levels of nitric oxide (NO) in the blood-filled midgut that adversely impact parasite development. In mammals, P. falciparum glycosylphosphatidylinositols (PfGPIs) can induce NOS expression in immune and endothelial cells and are sufficient to reproduce the major effects of parasite infection. These effects are mediated in part by mimicry of insulin signaling by PfGPIs. In this study, we demonstrate that PfGPIs can induce AsNOS expression in A. stephensi cells in vitro and in the midgut epithelium in vivo. Signaling by P. falciparum merozoites and PfGPIs is mediated through A. stephensi Akt/protein kinase B and a pathway involving DSOR1, a mitogen-activated protein kinase kinase, and an extracellular signal-regulated kinase. However, despite the involvement of kinases that are also associated with insulin signaling in A. stephensi cells, signaling by P. falciparum and by PfGPIs is distinctively different from signaling by insulin. Therefore, although mimicry of insulin by PfGPIs appears to be restricted to mammalian hosts of P. falciparum, the conservation of PfGPIs as a prominent parasite-derived signal of innate immunity can now be extended to include Anopheles mosquitoes, indicating that parasite signaling of innate immunity is conserved in mosquito and mammalian cells.     

Effect of insulin-like growth factor-I on Leishmania amazonensis promastigote arginase activation and reciprocal inhibition of NOS2 pathway in macrophage in vitro

We showed previously that insulin-like growth factor (IGF)-I induces an exacerbation of the lesion development in experimental cutaneous leishmaniasis favouring parasite growth within host macrophages. Here we studied the effect of IGF-I in vitro in BALB/c mouse peritoneal macrophages infected with stationary phase Leishmania amazonensis promastigotes. IGF-I was used to pre-incubate either macrophage or parasite before infection of the macrophages or adding it at the start of the Leishmania-macrophage culture and maintaining it throughout the experimental period. Independent of stimulation protocol, IGF-I induced significantly increased parasite growth within macrophages. Arginase activation considered as a key factor in Leishmania growth was studied, and its expression and activity were increased in Leishmania-infected macrophages but significantly more in infected cells upon IGF-I stimulus, an effect specifically inhibited by NOHA. Arginase known to be present on Leishmania was also studied, and its expression and activity were seen in the absence of any stimulus but significantly increased after 5 min of incubation with IGF-I. In addition, Leishmania was pre-incubated with NOHA for 5 min, washed, then macrophages infected observing a significantly reduced parasite burden in both IGF-I-stimulated and non-stimulated macrophages. Reciprocal decrease in the nitric oxide (NO) level and inhibition of nitric oxide synthase (NOS2) expression were also observed in IGF-I-stimulated infected macrophages. Our data strongly suggest that IGF-I induces preferential expression and activation of Leishmania promastigote arginase, contributes to the alternative activation of macrophages in the context of innate immunity and interferes with NOS pathway in infected macrophages probably as a reciprocal effect.     

Malnutrition Alters the Innate Immune Response and Increases Early Visceralization following Leishmania donovani Infection

Malnutrition is a risk factor for the development of visceral leishmaniasis. However, the immunological basis for this susceptibility is unknown. We have developed a mouse model to study the effect of malnutrition on innate immunity and early visceralization following Leishmania donovani infection. Three deficient diets were studied, including 6, 3, or 1% protein; these diets were also deficient in iron, zinc, and calories. The control diet contained 17% protein, was zinc and iron sufficient, and was provided ab libitum. Three days after infection with L. donovani promastigotes, the total extradermal (lymph nodes, liver, and spleen) and skin parasite burdens were equivalent in the malnourished (3% protein) and control mice, but in the malnourished group, a greater percentage (39.8 and 4.0%, respectively; P = 0.009) of the extradermal parasite burden was contained in the spleen and liver. The comparable levels of parasites in the footpads in the two diet groups and the higher lymph node parasite burdens in the well-nourished mice indicated that the higher visceral parasite burdens in the malnourished mice were not due to a deficit in local parasite killing but to a failure of lymph node barrier function. Lymph node cells from the malnourished, infected mice produced increased levels of prostaglandin E(2) (PGE(2)) and decreased levels of interleukin-10. Inducible nitric oxide synthase activity was significantly lower in the spleen and liver of the malnourished mice. Thus, malnutrition causes a failure of lymph node barrier function after L. donovani infection, which may be related to excessive production of PGE(2) and decreased levels of IL-10 and nitric oxide.     

Dual role of monocyte‐derived dendritic cells in Trypanosoma cruzi infection

Pathogens can cause inflammation when inoculated into the skin. The vector‐transmitted protozoan parasite Trypanosoma cruzi induces poor cellular‐infiltration and disseminates, causing high mortality in the experimental model. Here, we characterized the inflammatory foci at the parasite inoculation site and secondary lymphoid organs using a murine model. While no macrophages and few neutrophils and monocytes (Mo) were recruited into the skin, T. cruzi infection elicited the mobilization of Ly6C⁺ Mo to draining lymph nodes and spleen. Over time, this population became enriched in CD11b⁺Ly6C⁺CD11c⁺MHCII⁺CD86⁺ cells resembling inflammatory dendritic cells (DCs). Adoptive transfer of Ly6C⁺ Mo purified from the bone marrow of CD11c‐GFP transgenic mice confirmed the monocytic origin of Ly6C⁺ DCs found in the spleen of infected animals. Isolated Mo‐derived cells not only produced TNF‐α and nitric oxide, but also IL‐10 and displayed a poor capacity to induce lymphoproliferation. Ablation of Mo‐derived cells by 5‐fluorouracil confirmed their dual role during infection, limiting the parasite load by inducible nitric oxide synthase‐related mechanisms and negatively affecting the development of anti‐parasite T‐cell response. This study demonstrated that consistent with their antagonistic properties, these cells not only control the parasite spreading but also its persistence in the host.     

Babesia bovis-stimulated macrophages express interleukin-1beta, interleukin-12, tumor necrosis factor alpha, and nitric oxide and inhibit parasite replication in vitro

The tick-transmitted hemoparasite Babesia bovis causes an acute infection that results in persistence and immunity against challenge infection in cattle that control the initial parasitemia. Resolution of acute infection with this protozoal pathogen is believed to be dependent on products of activated macrophages (Mphi), including inflammatory cytokines and nitric oxide (NO) and its derivatives. B. bovis stimulates inducible nitric oxide synthase (iNOS) and production of NO in bovine Mphi, and chemical donors of NO inhibit the growth of B. bovis in vitro. However, the induction of inflammatory cytokines in Mphi by babesial parasites has not been described, and the antiparasitic activity of NO produced by B. bovis-stimulated Mphi has not been definitively demonstrated. We report that monocyte-derived Mphi activated by B. bovis expressed enhanced levels of inflammatory cytokines interleukin-1beta (IL-1beta), IL-12, and tumor necrosis factor alpha that are important for stimulating innate and acquired immunity against protozoal pathogens. Furthermore, a lipid fraction of B. bovis-infected erythrocytes stimulated iNOS expression and NO production by Mphi. Cocultures of Mphi and B. bovis-infected erythrocytes either in contact or physically separated resulted in reduced parasite viability. However, NO produced by bovine Mphi in response to B. bovis-infected erythrocytes was only partially responsible for parasite growth inhibition, suggesting that additional factors contribute to the inhibition of B. bovis replication. These findings demonstrate that B. bovis induces an innate immune response that is capable of controlling parasite replication and that could potentially result in host survival and parasite persistence.     

Inducible nitric oxide synthase deficiency in mice increases resistance to chronic infection with Echinococcus multilocularis

The production of nitric oxide (NO) by intraperitoneal macrophages of mice during secondary infection with Echinococcus multilocularis mediates immunosuppression at early and late stages of infection. We addressed the role of NO in host resistance against this extracellular metazoan parasite by infecting inducible nitric oxide synthase knockout ((iNOS-KO) mice (of the C57BL/6 background) with 100 metacestode vesicles. The parasite weight was significantly lower in iNOS-KO mice when compared with wild-type (WT) mice at 4 months postinfection (late stage), thus demonstrating that iNOS deficiency confers a certain degree of resistance against persistent chronic infection. However, histological analysis of periparasitic tissue showed no differences between WT and iNOS-KO mice, as both exhibited granuloma formation and the presence of giant cells. Together with histology, the production of a high level of interferon-gamma (IFN-gamma) in infected iNOS-KO mice upon stimulation with concanavalin A (Con A) and VF-antigen indicated normal T-cell signalling in these animals. As expected, peritoneal exudate cells (PEC) from infected iNOS-KO mice produced no detectable NO, while the PEC from infected WT mice produced high levels of NO after stimulation with lipopolysaccharide (LPS) and parasite protein or carbohydrate antigen, or even without in vitro stimulation. Consequently, the high level of NO production observed during chronic infection in WT mice appears to contribute more to immunosuppression than to limitation of parasite growth. This is also reflected by the fact that splenocyte proliferation was significantly higher and parasite masses lower in iNOS-KO mice (at 1 and 4 months postinfection) than in WT mice.     

Qualitative and quantitative immunohistochemical evaluation of iNOS expression in the spleen of dogs naturally infected with Leishmania chagasi

Nitric oxide (NO), the product of the nitric oxide synthase enzymes has been detected in Leishmania-infected animals. Besides its role on the immunity to infection, the role of NO and the inducible nitric oxide synthase (iNOS) in the pathogenesis of canine visceral leishmaniasis (CVL) is not well understood. This study aimed at evaluating immunohistochemically the iNOS expression in the spleen of dogs naturally infected (ID) with Leishmania (L.) chagasi compared with non-infected dogs (NID). The ID was grouped according to the clinical form and the parasite load. Symptomatic dogs (SD) presented higher parasite load in relation to oligosymptomatic (OD) and asymptomatic (AD). The qualitative expression of iNOS was observed only in ID. SD presented strong and prominent labeling of iNOS, followed by OD and AD. Quantitatively, the results showed that the median expression of iNOS was higher in SD and OD compared to NID. Also, dog spleens with high parasitism load showed marked iNOS expression. Taken together, the results suggest that the expression of iNOS in the spleen of infected dogs with CVL was associated with clinical worsening of the disease and with high parasitism.     

Role of gamma interferon in cellular immune response against murine Encephalitozoon cuniculi infection

Microsporidia are obligate intracellular protozoan parasites that cause a wide variety of opportunistic infection in patients with AIDS. Because it is able to grow in vitro, Encephalitozoon cuniculi is currently the best-studied microsporidian. T cells mediate protective immunity against this parasite. Splenocytes obtained from infected mice proliferate in vitro in response to irradiated parasites. A transient state of hyporesponsiveness to parasite antigen and mitogen was observed at day 17 postinfection. This downregulatory response could be partially reversed by addition of nitric oxide (NO) antagonist to the culture. Mice infected with E. cuniculi secrete significant levels of gamma interferon (IFN-gamma). Treatment with antibody to IFN-gamma or interleukin-2 (IL-12) was able to neutralize the resistance to the parasite. Mutant animals lacking the IFN-gamma or IL-12 gene were highly susceptible to infection. However, mice unable to secrete NO withstood high doses of parasite challenge, similar to normal wild-type animals. These studies describe an IFN-gamma-mediated protection against E. cuniculi infection that is independent of NO production.     

Effective clearance of intracellular Leishmania major in vivo requires Pten in macrophages

Leishmaniases are a major international public health problem, and macrophages are crucial for host resistance to this parasite. To determine if phosphatase and tensin homologue deleted on chromosome ten (Pten), a negative regulator of the PI3K pathway, plays a role in macrophage-mediated resistance to Leishmania, we generated C57BL/6 mice lacking Pten specifically in macrophages (LysMCrePten(flox/flox) mice). Examination of lesions resulting from Leishmania major infection showed that LysMCrePten(flox/flox) mice were more susceptible to the parasite than wild-type (WT) mice in the early phase of the infection, but were eventually able to eliminate the pathogen. In vitro Pten-deficient macrophages showed a reduced ability to kill parasites in response to IFN-gamma treatment, possibly because the mutant cells exhibited decreased TNF secretion that correlated with reductions in inducible nitric oxide synthase expression and nitric oxide production. In response to various TLR ligands, Pten-deficient macrophages produced less TNF and IL-12 but more IL-10 than WT cells. However, analysis of cells in the lymph nodes draining L. major inoculation sites indicated that both LysMCrePten(flox/flox) and WT mice developed normal Th1 responses following L. major infection, in line with the ability of LysMCrePten(flox/flox) mice to eventually eliminate the parasite. Our results indicate that the efficient clearance of intracellular parasites requires Pten in macrophages.     

Nitric oxide production by Leishmania-infected macrophages and modulation by prostaglandin E2

Nitric oxide (NO), produced by the nitric oxide synthase (iNOS) enzyme, is the most-important molecule responsible for the killing of Leishmania parasites by macrophages. In previous work we have demonstrated that, after activation with recombinant human interferon-γ and/or bacterial lipopolysaccharide, human macrophages infected with Leishmania infantum are able to produce nitric oxide and to express nitric oxide synthase. The arachidonate derivative prostaglandin E₂ has been shown to modulate various macrophage activities, and in particular nitric oxide production, sometimes with opposite effects, related to experimental conditions. In this work we have evaluated nitric oxide release and parasite killing by peripheral blood-derived L. infantum-infected human macrophages in vitro stimulated with lipopolysaccharide and simultaneously treated with prostaglandin E₂. Experiments were also performed in the presence of the nitric oxide synthase inhibitor l-N ^G monomethylarginine (l-NMMA) and of the cyclooxygenase inhibitor indomethacin. Nitric oxide release in supernatants of macrophage cultures was measured by the Griess reaction for nitrites. Parasite killing was microscopically evaluated by fluorescent dyes. Results demonstrated that macrophages stimulated with lipopolysaccharide and treated with prostaglandin E₂ exhibited increased nitric oxide producation and parasite killing, which were significantly reduced by either l-NMMA or indomethacin. In indomethacin-treated macrophages, nitric oxide production and leishmanicidal ability were partially restored by the addition of exogenous prostaglandin E₂. Taken together, these results indicate that prostaglandin E₂ may be involved in nitric oxide production, and possibly in the host-protective immune response against Leishmania. Moreover, the demonstration of a stimulatory role of prostaglandin E₂ on nitric oxide production induced by intracellular pathogens in humans is interesting in the light of a possible pharmacological regulation of nitric oxide by modulation of prostaglandin E₂ synthesis. Received: 27 March 2001 / Accepted: 24 August 2001     

The efficacy of enrofloxacin,alone or combined with metronidazole,in the therapy of canine leishmaniasis

We evaluated the efficacy of enrofloxacin, alone or combined with metronidazole, against Leishmania infantum. The in vitro activity of this fluoroquinolone was assessed using two different methods: a direct test aimed at assessing the drug activity on the parasite, and an indirect test aimed at evaluating the drug effect on macrophage killing, lymphomonocyte activation and nitric oxide production. An in vivo test was also performed on 36 dogs with leishmaniasis, subdivided into three groups, one treated with enrofloxacin, another with enrofloxacin plus metronidazole, and a control group with meglumine antimoniate. The direct test did not show any action of enrofloxacin on the parasite, while the indirect testing showed an enhancement of macrophage killing and an increase in nitric oxide production. These findings show that enrofloxacin does not exert a direct anti-leishmanial activity in vitro. However, on the basis of the positive immunostimulation results shown in vitro and the clinical improvement, particularly of the cutaneous lesions, obtained in several dogs in the in vivo trial, the use of enrofloxacin in association with a specific anti-leishmanial drug can be proposed in the therapeutic protocol of canine leishmaniasis.     

Malaria: becoming more specific about non-specific immunity.

For the hundreds of millions of people presently infected with malaria, survival may depend on relatively non-specific immune effector mechanisms. Progress has been made in understanding the anti-parasitic properties of tumor necrosis factor-alpha, interferon-gamma and nitric oxide, in defining the parasite toxins that induce tumor necrosis factor-alpha production, and in exploring the role of cytokines and adhesion molecules in the pathogenesis of cerebral malaria.     

Purification of Plasmodium falciparum digestive vacuoles and partial characterization of the vacuolar membrane ATPase

Plasmodium falciparum digestive vacuoles containing ferric oxide granules were purified from parasite homogenates by centrifugation on discontinuous sucrose gradients. Digestive vacuole membranes prepared by osmotic lysis and washed with KCl showed no detectable contamination by erythrocyte membrane proteins and only minimal contamination by non-vacuolar parasite proteins. Purified vacuolar membranes were 2.6-fold enriched in total parasite membrane ATPase activity. This ATPase was optimally active at pH 7 in the presence of at least 2 mM Mg2+. Ca2+ and Mn2+ were approximately 80-90% as effective as Mg2+, and Zn2+, Co2+ and Fe2+ also exerted some stimulatory effect. The vacuolar membrane also hydrolyzed GTP, UTP, CTP and ADP, but AMP and 3',5'-cyclic AMP were hydrolyzed only one-tenth as effectively as ATP. The ATPase was unaffected by vanadate, ouabain or oligomycin but was significantly inhibited by the proton pump inhibitors NEM and NBD-Cl. Of 6 antimalarial drugs tested, quinine and quinacrine were the most effective inhibitors and mefloquine was the least effective.