Leishmania Promastigotes Enhance Neutrophil Recruitment through the Production of CXCL8 by Endothelial Cells.

Endothelial cells represent one of the first cell types encountered by Leishmania promastigotes when inoculated into the skin of the human hosts by the bite of phlebotomine sand flies. However, little is known on their role in the early recruitment of phagocytic cells and in the establishment of the infection. Initially, neutrophils, rapidly recruited to the site of promastigotes deposition, phagocytize Leishmania promastigotes, which elude the killing mechanisms of the host cells, survive, and infect other phagocytic cells. Here, we show that Leishmania promastigotes co-incubated with HMEC-1, a microvascular endothelial cell line, exhibited significant morphological changes and loss of infectivity. Moreover, promastigotes of different Leishmania species stimulated the production of CXCL8 by HMEC-1 in a dose- and TLR4-dependent manner. Interestingly, we observed that the conditioned media from Leishmania-stimulated HMEC-1 cells attracted leukocytes, mostly neutrophils, after 2 h of incubation. After 24 h, a higher percentage of monocytes was detected in conditioned media of unstimulated HMEC-1 cells, whereas neutrophils still predominated in conditioned medium from Leishmania-stimulated cells. The same supernatants did not contain CCL5, a chemokine recruiting T cells and monocytes. On the contrary, inhibition of the production of CCL5 induced by TNF-α was seen. These data indicate that the interaction of Leishmania promastigotes with endothelial cells leads to the production of chemokines and the recruitment of neutrophils, which contribute to the establishment of Leishmania infection.

Phagocytosis and activation of bone marrow-derived macrophages by Plasmodium falciparum gametocytes.

BACKGROUND: The innate immune response against various life cycle stages of the malaria parasite plays an important role in protection against the disease and regulation of its severity. Phagocytosis of asexual erythrocytic stages is well documented, but little and contrasting results are available about phagocytic clearance of sexual stages, the gametocytes, which are responsible for the transmission of the parasites from humans to mosquitoes. Similarly, activation of host macrophages by gametocytes has not yet been carefully addressed. METHODS: Phagocytosis of early or late Plasmodium falciparum gametocytes was evaluated through methanol fixed cytospin preparations of immortalized mouse C57Bl/6 bone marrow-derived macrophages treated for 2 h with P. falciparum and stained with Giemsa, and it was confirmed through a standardized bioluminescent method using the transgenic P. falciparum 3D7elo1-pfs16-CBG99 strain. Activation was evaluated by measuring nitric oxide or cytokine levels in the supernatants of immortalized mouse C57Bl/6 bone marrow-derived macrophages treated with early or late gametocytes. RESULTS: The results showed that murine bone marrow-derived macrophages can phagocytose both early and late gametocytes, but only the latter were able to induce the production of inflammatory mediators, specifically nitric oxide and the cytokines tumour necrosis factor and macrophage inflammatory protein 2. CONCLUSIONS: These results support the hypothesis that developing gametocytes interact in different ways with innate immune cells of the host. Moreover, the present study proposes that early and late gametocytes act differently as targets for innate immune responses.

Boosting immunity to treat parasitic infections: Asaia bacteria expressing a protein from Wolbachia determine M1 macrophage activation and killing of Leishmania protozoans.

Leishmaniases are severe vector-borne diseases affecting humans and animals, caused by Leishmania protozoans. Over one billion people and millions of dogs live in endemic areas for leishmaniases and are at risk of infection. Immune polarization plays a major role in determining the outcome of Leishmania infections: hosts displaying M1-polarized macrophages are protected, while those biased on the M2 side acquire a chronic infection that could develop into a deadly disease. The identification of the factors involved in M1 polarization is essential for the design of therapeutic and prophylactic interventions, including vaccines. Infection by the filarial nematode Dirofilaria immitis could be one of the factors that interfere with leishmaniasis in dogs. Indeed, filarial nematodes induce a partial skew of the immune response towards M1, likely caused by their bacterial endosymbionts, Wolbachia. Here we have examined the potential of AsaiaWSP, a bacterium engineered for the expression of the Wolbachia surface protein (WSP), as an inductor of M1 macrophage activation and Leishmania killing. Macrophages stimulated with AsaiaWSP displayed a strong leishmanicidal activity, comparable to that determined by the choice-drug amphotericin B. Additionally, AsaiaWSP determined the expression of markers of classical macrophage activation, including M1 cytokines, ROS and NO, and an increase in phagocytosis activity. Asaia not expressing WSP also induced macrophage activation, although at a lower extent compared to AsaiaWSP. In summary, the results of the present study confirm the immunostimulating properties of WSP highlighting a potential therapeutic efficacy against Leishmania parasites. Furthermore, Asaia was designed as a delivery system for WSP, thus developing a novel type of immunomodulating agent, worthy of being investigated for immuno-prophylaxis and -therapy of leishmaniases and other diseases that could be subverted by M1 macrophage activation.

Effects of combined drug treatments on Plasmodium falciparum: In vitro assays with doxycycline, ivermectin and efflux pump inhibitors.

There is great concern regarding the rapid emergence and spread of drug-resistance in Plasmodium falciparum, the parasite responsible for the most severe form of human malaria. Parasite populations resistant to some or all the currently available antimalarial treatments are present in different world regions. Considering the need for novel and integrated approaches to control malaria, combinations of drugs were tested on P. falciparum. The primary focus was on doxycycline, an antibiotic that specifically targets the apicoplast of the parasite. In combination with doxycycline, three different drugs known to inhibit efflux pumps (verapamil, elacridar and ivermectin) were tested, with the assumption that they could increase the intracellular concentration of the antibiotic and consequently its efficacy against P. falciparum. We emphasize that elacridar is a third-generation ABC transporters inhibitor, never tested before on malaria parasites. In vitro experiments were performed on asexual stages of two strains of P. falciparum, chloroquine-sensitive (D10) and chloroquine-resistant (W2). Incubation times on asynchronous or synchronous cultures were 72h or 96h, respectively. The antiplasmodial effect (i.e. the IC50) was determined by measuring the activity of the parasite lactate dehydrogenase, while the interaction between drugs was determined through combination index (CI) analyses. Elacridar achieved an IC50 concentration comparable to that of ivermectin, approx. 10-fold lower than that of verapamil, the other tested ABC transporter inhibitor. CI results showed synergistic effect of verapamil plus doxycycline, which is coherent with the starting hypothesis, i.e. that ABC transporters represent potential targets, worth of further investigations, towards the development of companion molecules useful to enhance the efficacy of antimalarial drugs. At the same time, the observed antagonistic effect of doxycycline in combination with ivermectin or elacridar highlighted the importance of drug testing, to avoid the de-facto generation of a sub-dosage, a condition that facilitates the development of drug resistance.

Malaria pigment accelerates MTT - formazan exocytosis in human endothelial cells.

Haemozoin is a by-product of haemoglobin digestion by intraerythrocytic malaria parasites, which induces immunologic responses on different tissues, including endothelial cells. In the present paper, the incubation of human microvascular endothelial cells with haemozoin significantly inhibited MTT reduction, a measure of cytotoxicity, without increasing the release of cytoplasmic lactate dehydrogenase. Moreover, haemozoin did not induce apoptosis or cell cycle arrest nor decreased the number of live cells, suggesting that cells viability itself was not affected and that the inhibition of MTT reduction was only apparent and probably due to accelerated MTT-formazan exocytosis. After 30 min of MTT addition, a significant increase in the % of cells exocytosing MTT formazan crystals was observed in haemozoin-treated cells compared with control cells. Such an effect was partially reversed by the addition of genistein, an inhibitor of MTT-formazan exocytosis. The rapid release of CXCL-8, a preformed chemokine contained in Weibel-Palade bodies, confirmed that haemozoin induces a perturbation of the intracellular endothelial trafficking, including the exocytosis of MTT-formazan containing vesicles. The haem moiety of haemozoin is responsible for the observed effect. Moreover, this work underlines that MTT assay should not be used to measure cytotoxicity induced by haemozoin and other methods should be preferred.

Interplay between Plasmodium falciparum haemozoin and L-arginine: implication for nitric oxide production.

BACKGROUND: Plasmodium falciparum haemozoin, a detoxification product of digested haemoglobin from infected erythrocytes, is released into the bloodstream upon schizont rupture and accumulates in leukocytes. High levels of haemozoin correlate with disease severity. Some studies have shown that concentrations of the substrate of inducible nitric oxide synthase (iNOS), L-arginine, as well as nitric oxide are low in patients infected with P. falciparum malaria. The present study investigates, in vitro, the role of P. falciparum haemozoin on nitric oxide production, iNOS expression in macrophages, and the possible interaction between L-arginine and haemozoin. METHODS: Plasmodium falciparum haemozoin was obtained from in vitro cultures through magnetic isolation. Phagocytosis of haemozoin by immortalized bone marrow derived macrophages was detected by confocal reflection combined with fluorescence microscopy. Nitrite concentrations in the supernatants was evaluated by Griess assay as a standard indication of nitric oxide production, while iNOS expression was detected on cell extracts by western blotting. Detection of L-arginine in haemozoin-treated or untreated media was achieved by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: Haemozoin synergizes in vitro with interferon-gamma to produce nitric oxide. However, when mouse macrophages were stimulated with haemozoin, a proportional increase of nitric oxide was observed up to 25 µM of haemozoin, followed by a decrease with doses up to 100 µM, when nitric oxide release was completely abrogated. This was not due to reactive oxygen species production, nor to an effect on iNOS activity. Interestingly, when at 24 h, haemozoin-treated macrophages were washed and incubated in fresh medium for further 24 h, the nitric oxide production was restored in a dose-response manner. Similar results were seen when L-arginine-enriched media was used in the stimulation. Moreover, muramyldipeptide, a strong nitric oxide inducer, was unable to activate macrophages to release nitric oxide in the presence of haemozoin-treated medium. By LC-MS/MS a complete depletion of L-arginine was observed in this haemozoin-treated, conditioned medium. CONCLUSIONS: It is proposed that haemozoin interacts with L-arginine reducing its availability for iNOS, and thus decreasing nitric oxide production. The clinical (or pathological) implications of these results are discussed.

Differential induction of malaria liver pathology in mice infected with Plasmodium chabaudi AS or Plasmodium berghei NK65.

BACKGROUND: Cerebral malaria and severe anaemia are the most common deadly complications of malaria, and are often associated, both in paediatric and adult patients, with hepatopathy, whose pathogenesis is not well characterized, and sometimes also with acute respiratory distress syndrome (ARDS). Here, two species of murine malaria, the lethal Plasmodium berghei strain NK65 and self-healing Plasmodium chabaudi strain AS which differ in their ability to cause hepatopathy and/or ARDS were used to investigate the lipid alterations, oxidative damage and host immune response during the infection in relation to parasite load and accumulation of parasite products, such as haemozoin. METHODS: Plasma and livers of C57BL/6J mice injected with PbNK65 or PcAS infected erythrocytes were collected at different times and tested for parasitaemia, content of haemozoin and expression of tumour necrosis factor (TNF). Hepatic enzymes, antioxidant defenses and lipids content and composition were also evaluated. RESULTS: In the livers of P. berghei NK65 infected mice both parasites and haemozoin accumulated to a greater extent than in livers of P. chabaudi AS infected mice although in the latter hepatomegaly was more prominent. Hepatic enzymes and TNF were increased in both models. Moreover, in P. berghei NK65 infected mice, increased lipid peroxidation, accumulation of triglycerides, impairment of anti-oxidant enzymes and higher collagen deposition were detected. On the contrary, in P. chabaudi AS infected mice the antioxidant enzymes and the lipid content and composition were normal or even lower than uninfected controls. CONCLUSIONS: This study demonstrates that in C57BL/6J mice, depending on the parasite species, malaria-induced liver pathology results in different manifestations, which may contribute to the different outcomes. In P. berghei NK65 infected mice, which concomitantly develop lethal acute respiratory distress syndrome, the liver tissue is characterized by an excess oxidative stress response and reduced antioxidant defenses while in P. chabaudi AS infected mice hepatopathy does not lead to lipid alterations or reduction of antioxidant enzymes, but rather to inflammation and cytokine burst, as shown earlier, that may favour parasite killing and clearance of the infection. These results may help understanding the different clinical profiles described in human malaria hepatopathy.

Adenosine Triphosphate-Binding Cassette Transporters Are Not Involved In the Detoxification of Azadirachta indica Extracts In Anopheles stephensi Larvae.

Detoxifying pathways of mosquitoes against the neem (Azadirachta indica) extracts are still unclear. The aim of the present study was to investigate the role of adenosine triphosphate-binding cassette (ABC) transporters in this process in Anopheles stephensi, one of the main malaria vectors in southern Asia. Third-stage larvae of An. stephensi were fed with fish food alone or in combination with neem extract at 0.5%, 1%, 5%, and 10%. Six ABC-transporter genes from 3 different subfamilies (B, C, and G) were analyzed to assess their relative expression compared with controls. A bioassay was also performed to assess larval mortality rate at different concentrations and in combination with verapamil, an ABC-transporter inhibitor. No significant variation in the expression levels of any transporter belonging to the B, C, and G subfamilies was detected. Furthermore, the use of verapamil did not induce an increase in mortality at any of the tested neem extract concentrations, indicating that ABC transporters are not involved in the detoxification of neem extracts in An. stephensi larvae.

Malaria pigment stimulates chemokine production by human microvascular endothelium.

Severe falciparum malaria is characterized by the sequestration of infected erythrocytes and leukocyte recruitment in the microvasculature, resulting in impaired blood flow and metabolic disturbances. Which parasite products cause chemokine production, thus contributing to the strong host inflammatory response and cellular recruitment are not well characterized. Here, we studied haemozoin (Hz), the end-product of haem, a ferriprotoporphyrin-IX crystal bound to host and parasite lipids, DNA, and proteins. We found that natural Hz isolated from Plasmodium falciparum cultures induces CXCL8 and CCL5 production in human dermal microvascular endothelial cells (HMEC-1) in a time-dependent manner. This up-regulation is not caused by haem but rather by Hz-generated lipoperoxidation products (15-HETE) and fibrinogen associated to Hz, and is, at least in part, triggered by the activation of NF-κB, as it was significantly inhibited by artemisinin and other NF-κB pathway inhibitors.

HaCaT Cells as a Reliable In Vitro Differentiation Model to Dissect the Inflammatory/Repair Response of Human Keratinocytes.

Cultured primary human keratinocytes are frequently employed for studies of immunological and inflammatory responses; however, interpretation of experimental data may be complicated by donor to donor variability, the relatively short culture lifetime, and variations between passages. To standardize the in vitro studies on keratinocytes, we investigated the use of HaCaT cells, a long-lived, spontaneously immortalized human keratinocyte line which is able to differentiate in vitro, as a suitable model to follow the release of inflammatory and repair mediators in response to TNFα or IL-1ß. Different treatment conditions (presence or absence of serum) and differentiation stimuli (increase in cell density as a function of time in culture and elevation of extracellular calcium) were considered. ELISA and Multiplex measurement technologies were used to monitor the production of cytokines and chemokines. Taken together, the results highlight that Ca2+ concentration in the medium, cell density, and presence of serum influences at different levels the release of proinflammatory mediators by HaCaT cells. Moreover, HaCaT cells maintained in low Ca2+ medium and 80% confluent are similar to normal keratinocytes in terms of cytokine production suggesting that HaCaT cells may be a useful model to investigate anti-inflammatory interventions/therapies on skin diseases.

Open Source Drug Discovery with the Malaria Box Compound Collection for Neglected Diseases and Beyond.

A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts.

A chemical susceptibility profile of the Plasmodium falciparum transmission stages by complementary cell-based gametocyte assays.

OBJECTIVES: As most available antimalarial drugs are ineffective against the Plasmodium falciparum transmission stages, new drugs against the parasite's gametocytes are urgently needed to combat malaria globally. The unique biology of gametocytes requires assays that need to be specific, to faithfully monitor anti-gametocyte activity, and to be easy to perform, cheap and scalable to high-throughput screening (HTS). METHODS: We developed an HTS cell-based assay with P. falciparum gametocytes specifically expressing a potent luciferase. To confirm HTS hit activity for several parasite genotypes, the luciferase assay and the gametocyte lactate dehydrogenase (LDH) assay, usable on any parasite isolate, were compared by screening antimalarial drugs and determining IC50 values of anti-gametocyte hits from the 'Malaria Box' against early- and late-stage gametocytes. RESULTS: Comparison of the two assays, conducted on the early and on late gametocyte stages, revealed an excellent correlation (R(2) > 0.9) for the IC50 values obtained by the respective readouts. Differences in susceptibility to drugs and compounds between the two parasite developmental stages were consistently measured in both assays. CONCLUSIONS: This work indicates that the luciferase and gametocyte LDH assays are interchangeable and that their specific advantages can be exploited to design an HTS pipeline leading to new transmission-blocking compounds. Results from these assays consistently defined a gametocyte chemical susceptibility profile, relevant to the planning of future drug discovery strategies.

Salinomycin and other ionophores as a new class of antimalarial drugs with transmission-blocking activity.

The drug target profile proposed by the Medicines for Malaria Venture for a malaria elimination/eradication policy focuses on molecules active on both asexual and sexual stages of Plasmodium, thus with both curative and transmission-blocking activities. The aim of the present work was to investigate whether the class of monovalent ionophores, which includes drugs used in veterinary medicine and that were recently proposed as human anticancer agents, meets these requirements. The activity of salinomycin, monensin, and nigericin on Plasmodium falciparum asexual and sexual erythrocytic stages and on the development of the Plasmodium berghei and P. falciparum mosquito stages is reported here. Gametocytogenesis of the P. falciparum strain 3D7 was induced in vitro, and gametocytes at stage II and III or stage IV and V of development were treated for different lengths of time with the ionophores and their viability measured with the parasite lactate dehydrogenase (pLDH) assay. The monovalent ionophores efficiently killed both asexual parasites and gametocytes with a nanomolar 50% inhibitory concentration (IC50). Salinomycin showed a fast speed of kill compared to that of standard drugs, and the potency was higher on stage IV and V than on stage II and III gametocytes. The ionophores inhibited ookinete development and subsequent oocyst formation in the mosquito midgut, confirming their transmission-blocking activity. Potential toxicity due to hemolysis was excluded, since only infected and not normal erythrocytes were damaged by ionophores. Our data strongly support the downstream exploration of monovalent ionophores for repositioning as new antimalarial and transmission-blocking leads.

cAMP-Signalling Regulates Gametocyte-Infected Erythrocyte Deformability Required for Malaria Parasite Transmission.

Blocking Plasmodium falciparum transmission to mosquitoes has been designated a strategic objective in the global agenda of malaria elimination. Transmission is ensured by gametocyte-infected erythrocytes (GIE) that sequester in the bone marrow and at maturation are released into peripheral blood from where they are taken up during a mosquito blood meal. Release into the blood circulation is accompanied by an increase in GIE deformability that allows them to pass through the spleen. Here, we used a microsphere matrix to mimic splenic filtration and investigated the role of cAMP-signalling in regulating GIE deformability. We demonstrated that mature GIE deformability is dependent on reduced cAMP-signalling and on increased phosphodiesterase expression in stage V gametocytes, and that parasite cAMP-dependent kinase activity contributes to the stiffness of immature gametocytes. Importantly, pharmacological agents that raise cAMP levels in transmissible stage V gametocytes render them less deformable and hence less likely to circulate through the spleen. Therefore, phosphodiesterase inhibitors that raise cAMP levels in P. falciparum infected erythrocytes, such as sildenafil, represent new candidate drugs to block transmission of malaria parasites.

Involvement of Nod2 in the innate immune response elicited by malarial pigment hemozoin.

In malaria, the evidence concerning the nucleotide-binding, oligomerization domain (NOD) 2 (NOD2) receptor is fragmented and the stimuli that might activate NOD2 are not well characterized. We investigated the role of NOD2 in vitro in the response of macrophages to Plasmodium falciparum products. Immortalized or primary bone marrow derived macrophages from wild type C57Bl/6 mice, or knockout mice for NOD2 or its adaptor proteins, were either primed with interferon gamma or left untreated, and stimulated with parasite products. Both lysates of infected erythrocytes or hemozoin induced higher levels of nitric oxide in primed than in unprimed wild type macrophages. When stimulated with hemozoin, primed macrophages knockout for NOD2, or for its adaptor proteins, produced significantly lower nitric oxide levels compared to wild type cells. Differently from hemozoin, the use of ß-hematin (synthetic hemozoin) as stimulus showed that NOD2 is dispensable. Furthermore, the production of inflammatory cytokines by wild type cells treated with hemozoin was not dependent on NOD2. These data indicate that parasite components present in the hemozoin, differently from ß-hematin, induce the production of nitric oxide through the activation of NOD2, whereas the production of inflammatory cytokines, like TNF-α or MIP-2 (CXCL2), seems to be NOD2 independent.

Dual engagement of the NLRP3 and AIM2 inflammasomes by plasmodium-derived hemozoin and DNA during malaria.

Hemozoin (Hz) is the crystalline detoxification product of hemoglobin in Plasmodium-infected erythrocytes. We previously proposed that Hz can carry plasmodial DNA into a subcellular compartment that is accessible to Toll-like receptor 9 (TLR9), inducing an inflammatory signal. Hz also activates the NLRP3 inflammasome in primed cells. We found that Hz appears to colocalize with DNA in infected erythrocytes, even before RBC rupture or phagolysosomal digestion. Using synthetic Hz coated in vitro with plasmodial genomic DNA (gDNA) or CpG oligodeoxynucleotides, we observed that DNA-complexed Hz induced TLR9 translocation, providing a priming and an activation signal for inflammasomes. After phagocytosis, Hz and DNA dissociate. Hz subsequently induces phagolysosomal destabilization, allowing phagolysosomal contents access to the cytosol, where DNA receptors become activated. Similar observations were made with Plasmodium-infected RBCs. Finally, infected erythrocytes activated both the NLRP3 and AIM2 inflammasomes. These observations suggest that Hz and DNA work together to induce systemic inflammation during malaria.

Antiplasmodial and anti-inflammatory activities of Canthium henriquesianum (K. Schum), a plant used in traditional medicine in Burkina Faso.

ETHNOPHARMACOLOGICAL RELEVANCE: Canthium henriquesianum (K. Schum) is traditionally used in Burkina Faso for the treatment of malaria, but has not been properly investigated, yet. The aim of this study was to characterize in vitro the antiplasmodial and the anti-inflammatory activity of extracts from Canthium henriquesianum (K. Schum). In parallel, extracts of Gardenia sokotensis (Hutch) and Vernonia colorata (Willd), also traditionally used together in Burkina Faso and already reported with antimalarial activity, were compared. MATERIALS AND METHODS: Plant extracts were tested in vitro for antimalarial activity against chloroquine susceptible (D10) and resistant (W2) strains of Plasmodium falciparum using the lactate dehydrogenase assay. Cell cytotoxicity was assessed on human dermal fibroblast (HDF) by the MTT assay. The selectivity index (SI) was used as the ratio of the activity against the parasites compared to the toxicity of the plant extract against HDF. In vitro cytokine production was assessed by ELISA technique. RESULTS: Canthium henriquesianum aqueous extract had a moderate antimalarial activity (IC50<50 µg/ml) with a good selectivity index (SI=HDF/D10>7). Canthium henriquesianum diisopropyl ether extract was the most potent inhibitor of parasite growth with an IC50 9.5 µg/ml on W2 and 8.8 µg/ml on D10 and limited toxicity (SI>2). Gardenia sokotensis and Vernonia colorata aqueous extracts were shown to be significantly less active (IC50≥50 µg/ml) with substantial toxicity. In addition, when the production of IL-1ß and TNFα by lipopolysaccharide (LPS) or hemozoin (malaria pigment) stimulated human THP-1 monocytes was assayed, it was found that the extract of Canthium henriquesianum induced a dose-dependent inhibition of IL-1ß, but not of TNFα production, thus confirming its traditional use as antipyretic. By NMR analysis, the chromone was identified as the mostly represented compound in the diisopropyl ether extract of Canthium henriquesianum. Chromone however, was less active as antimalarial than the crude extract and it did not inhibit cytokine production at not toxic doses, indicating that other molecules in the total extracts contribute to the antiplasmodial and anti-inflammatory activity. CONCLUSION: Canthium henriquesianum seems to possess antimalarial activity in vitro and the ability to inhibit the production of the pyrogenic cytokine IL-1ß.

A Plasmodium falciparum screening assay for anti-gametocyte drugs based on parasite lactate dehydrogenase detection.

OBJECTIVES: Plasmodium gametocytes, responsible for malaria parasite transmission from humans to mosquitoes, represent a crucial target for new antimalarial drugs to achieve malaria elimination/eradication. We developed a novel colorimetric screening method for anti-gametocyte compounds based on the parasite lactate dehydrogenase (pLDH) assay, already standardized for asexual stages, to measure gametocyte viability and drug susceptibility. METHODS: Gametocytogenesis of 3D7 and NF54 Plasmodium falciparum strains was induced in vitro and asexual parasites were depleted with N-acetylglucosamine. Gametocytes were treated with dihydroartemisinin, epoxomicin, methylene blue, primaquine, puromycin or chloroquine in 96-well plates and the pLDH activity was evaluated using a modified Makler protocol. Mosquito infectivity was measured by the standard membrane feeding assay (SMFA). RESULTS: A linear correlation was found between gametocytaemia determined by Giemsa staining and pLDH activity. A concentration-dependent reduction in pLDH activity was observed after 72 h of drug treatment, whereas an additional 72 h of incubation without drugs was required to obtain complete inhibition of gametocyte viability. SMFA on treated and control gametocytes confirmed that a reduction in pLDH activity translates into reduced oocyst development in the mosquito vector. CONCLUSIONS: The gametocyte pLDH assay is fast, easy to perform, cheap and reproducible and is suitable for screening novel transmission-blocking compounds, which does not require parasite transgenic lines.

Pathogenesis of malaria in tissues and blood.

The clinical manifestations of severe malaria are several and occur in different anatomical sites. Both parasite- and host-related factors contribute to the pathogenicity of the severe forms of the disease. Cytoadherence of infected red blood cells to the vascular endothelium of different organs and rosetting are unique features of malaria parasites which are likely to contribute to the vascular damage and the consequent excessive inflammatory/immune response of the host. In addition to cerebral malaria or severe anaemia, which are quite common manifestation of severe malaria, clinical evidences of thrombocytopenia, acute respiratory distress syndrome (ARDS), liver and kidney disease, are reported. In primigravidae from endemic areas, life threatening placental malaria may also be present.In the following pages, some of the pathogenetic aspects will be briefly reviewed and then data on selected and less frequent manifestation of severe malaria, such as liver or renal failure or ARDS will be discussed.