A Novel Calcium-Dependent Protein Kinase 1 Inhibitor Potently Prevents Toxoplasma gondii Transmission to Foetuses in Mouse.

Treatments currently used to prevent congenital toxoplasmosis are non-specific of Toxoplasma gondii and have grievous side effects. To develop a more specific and less toxic drug, we have designed SP230, an imidazo[1,2-b]pyridazine salt targeting the Toxoplasma gondii calcium-dependent protein kinase 1 (TgCDPK1) and active against acute toxoplasmosis in mice. Efficiency of SP230 to inhibit foetal transmission of the parasite was evaluated in a mouse model of congenital toxoplasmosis. Swiss mice were infected at mid-pregnancy with tachyzoites or cysts of the ME49 strain of T. gondii by intraperitoneal and oral route, respectively, and treated with SP230 at 50 mg/kg for 5 days by the same routes. Parasite burden in organs of dams and in foetuses was measured by quantitative PCR. Intraperitoneal administration of SP230 drastically reduced the number of parasites (more than 97% of reduction) in the brain and lungs of dams, and led to a reduction of 66% of parasite burden in foetuses. Oral administration of SP230 was particularly efficient with 97% of reduction of parasite burdens in foetuses. SP230 did not impact number and weight of offspring in our conditions. This inhibitor of TgCDPK1 is a promising candidate for the development of alternative therapeutics to treat infected pregnant women.

Neospora caninum glycosylphosphatidylinositols used as adjuvants modulate cellular immune responses induced in vitro by a nanoparticle-based vaccine.

Neospora caninum causes abortion in ruminants, leading to important economic losses and no efficient treatment or vaccine against neosporosis is available. Considering the complexity of the strategies developed by intracellular apicomplexan parasites to escape immune system, future vaccine formulations should associate the largest panel of antigens and adjuvants able to better stimulate immune responses than natural infection. A mucosal vaccine, constituted of di-palmitoyl phosphatidyl glycerol-loaded nanoparticles (DGNP) and total extract (TE) of soluble antigens of Toxoplasma gondii, has demonstrated its efficacy, decreasing drastically the parasite burden. Here, DGNP were loaded with N. caninum TE and glycosylphosphatidylinositol (GPI) of N. caninum as Toll-like receptor (TLR) adjuvant able to induce specific cellular and humoral immune responses. Activation of TLR2 and TLR4 signalling pathway in HEK reporter cells induced by GPI was abrogated after its incorporation into DGNP. However, in murine bone marrow-derived dendritic cells, an adjuvant effect of GPI was observed with higher levels of interleukin (IL)-1ß, reduced levels of IL-6, IL-12p40 and IL-10, and decreased expression of major histocompatibility complex (MHC) molecules. GPI also modulated the responses of bovine peripheral blood mononuclear cells, by increasing the production of IFN-γ and by decreasing the expression of MHC molecules. Altogether, these results suggest that GPI delivered by the DGNP might modulate cell responses through the activation of an intracellular pathway of signalisation in a TLR-independent manner. In vivo experiments are needed to confirm the potent adjuvant properties of N. caninum GPI in a vaccine strategy against neosporosis.

Engineering and Functional Evaluation of Neutralizing Antibody Fragments Against Congenital Toxoplasmosis.

Maternal-fetal transmission of Toxoplasma gondii tachyzoites acquired during pregnancy has potentially dramatic consequences for the fetus. Current reference-standard treatments are not specific to the parasite and can induce severe side effects. In order to provide treatments with a higher specificity against toxoplasmosis, we developed antibody fragments-single-chain fragment variable (scFv) and scFv fused with mouse immunoglobulin G2a crystallizable fragment (scFv-Fc)-directed against the major surface protein SAG1. After validating their capacity to inhibit T. gondii proliferation in vitro, the antibody fragments' biological activity was assessed in vivo using a congenital toxoplasmosis mouse model. Dams were treated by systemic administration of antibody fragments and with prevention of maternal-fetal transmission being used as the parameter of efficacy. We observed that both antibody fragments prevented T. gondii dissemination and protected neonates, with the scFv-Fc format having better efficacy. These data provide a proof of concept for the use of antibody fragments as effective and specific treatment against congenital toxoplasmosis and provide promising leads.

The molecular targets of ivermectin and lotilaner in the human louse Pediculus humanus humanus: New prospects for the treatment of pediculosis.

Control of infestation by cosmopolitan lice (Pediculus humanus) is increasingly difficult due to the transmission of parasites resistant to pediculicides. However, since the targets for pediculicides have no been identified in human lice so far, their mechanisms of action remain largely unknown. The macrocyclic lactone ivermectin is active against a broad range of insects including human lice. Isoxazolines are a new chemical class exhibiting a strong insecticidal potential. They preferentially act on the γ-aminobutyric acid (GABA) receptor made of the resistant to dieldrin (RDL) subunit and, to a lesser extent on glutamate-gated chloride channels (GluCls) in some species. Here, we addressed the pediculicidal potential of isoxazolines and deciphered the molecular targets of ivermectin and the ectoparasiticide lotilaner in the human body louse species Pediculus humanus humanus. Using toxicity bioassays, we showed that fipronil, ivermectin and lotilaner are efficient pediculicides on adult lice. The RDL (Phh-RDL) and GluCl (Phh-GluCl) subunits were cloned and characterized by two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes. Phh-RDL and Phh-GluCl formed functional homomeric receptors respectively gated by GABA and L-glutamate with EC50 values of 16.0 µM and 9.3 µM. Importantly, ivermectin displayed a super agonist action on Phh-GluCl, whereas Phh-RDL receptors were weakly affected. Reversally, lotilaner strongly inhibited the GABA-evoked currents in Phh-RDL with an IC50 value of 40.7 nM, whereas it had no effect on Phh-GluCl. We report here for the first time the insecticidal activity of isoxazolines on human ectoparasites and reveal the mode of action of ivermectin and lotilaner on GluCl and RDL channels from human lice. These results emphasize an expected extension of the use of the isoxazoline drug class as new pediculicidal agents to tackle resistant-louse infestations in humans.

Molecular and Functional Characterization of GABA Receptor Subunits GRD and LCCH3 from Human Louse Pediculus Humanus Humanus.

Human louse Pediculus humanus is a cosmopolitan obligatory blood-feeding ectoparasite causing pediculosis and transmitting many bacterial pathogens. Control of infestation is difficult due to the developed resistance to insecticides that mainly target GABA (γ-aminobutyric acid) receptors. Previous work showed that Pediculus humanus humanus (Phh) GABA receptor subunit resistance to dieldrin (RDL) is the target of lotilaner, a synthetic molecule of the isoxazoline chemical class. To enhance our understanding of how insecticides act on GABA receptors, two other GABA receptor subunits were cloned and characterized: three variants of Phh-grd (glycine-like receptor of Drosophila) and one variant of Phh-lcch3 (ligand-gated chloride channel homolog 3). Relative mRNA expression levels of Phh-rdl, Phh-grd, and Phh-lcch3 revealed that they were expressed throughout the developmental stages (eggs, larvae, adults) and in the different parts of adult lice (head, thorax, and abdomen). When expressed individually in the Xenopus oocyte heterologous expression system, Phh-GRD1, Phh-GRD2, Phh-GRD3, and Phh-LCCH3 were unable to reconstitute functional channels, whereas the subunit combinations Phh-GRD1/Phh-LCCH3, Phh-GRD1/Phh-RDL, and Phh-LCCH3/Phh-RDL responded to GABA in a concentration-dependent manner. The three heteromeric receptors were similarly sensitive to the antagonistic effect of picrotoxin and fipronil, whereas Phh-GRD1/Phh-RDL and Phh-LCCH3/Phh-RDL were respectively about 2.5-fold and 5-fold more sensitive to ivermectin than Phh-GRD1/Phh-LCCH3. Moreover, the heteropentameric receptor constituted by Phh-GRD1/Phh-LCCH3 was found to be permeable and highly sensitive to the extracellular sodium concentration. These findings provided valuable additions to our knowledge of the complex nature of GABA receptors in human louse that could help in understanding the resistance pattern to commonly used pediculicides. SIGNIFICANCE STATEMENT: Human louse is an ectoparasite that causes pediculosis and transmits several bacterial pathogens. Emerging strains developed resistance to the commonly used insecticides, especially those targeting GABA receptors. To understand the molecular mechanisms underlying this resistance, two subunits of GABA receptors were cloned and described: Phh-grd and Phh-lcch3. The heteromeric receptor reconstituted with the two subunits was functional in Xenopus oocytes and sensitive to commercially available insecticides. Moreover, both subunits were transcribed throughout the parasite lifecycle.

Neospora caninum: a new class of biopharmaceuticals in the therapeutic arsenal against cancer.

BACKGROUND: Microorganisms that can be used for their lytic activity against tumor cells as well as inducing or reactivating antitumor immune responses are a relevant part of the available immunotherapy strategies. Viruses, bacteria and even protozoa have been largely explored with success as effective human antitumor agents. To date, only one oncolytic virus-T-VEC-has been approved by the US Food and Drug Administration for use in biological cancer therapy in clinical trials. The goal of our study is to evaluate the potential of a livestock pathogen, the protozoan Neospora caninum, non-pathogenic in humans, as an effective and safe antitumorous agent. METHODS/RESULTS: We demonstrated that the treatment of murine thymoma EG7 by subcutaneous injection of N. caninum tachyzoites either in or remotely from the tumor strongly inhibits tumor development, and often causes their complete eradication. Analysis of immune responses showed that N. caninum had the ability to 1) lyze infected cancer cells, 2) reactivate the immunosuppressed immune cells and 3) activate the systemic immune system by generating a protective antitumor response dependent on natural killer cells, CD8-T cells and associated with a strong interferon (IFN)-γ secretion in the tumor microenvironment. Most importantly, we observed a total clearance of the injected agent in the treated animals: N. caninum exhibited strong anticancer effects without persisting in the organism of treated mice. We also established in vitro and an in vivo non-obese diabetic/severe combined immunodeficiency mouse model that N. caninum infected and induced a strong regression of human Merkel cell carcinoma. Finally, we engineered a N. caninum strain to secrete human interleukin (IL)-15, associated with the alpha-subunit of the IL-15 receptor thus strengthening the immuno-stimulatory properties of N. caninum. Indeed, this NC1-IL15hRec strain induced both proliferation of and IFN-γ secretion by human peripheral blood mononuclear cells, as well as improved efficacy in vivo in the EG7 tumor model. CONCLUSION: These results highlight N. caninum as a potential, extremely effective and non-toxic anticancer agent, capable of being engineered to either express at its surface or to secrete biodrugs. Our work has identified the broad clinical possibilities of using N. caninum as an oncolytic protozoan in human medicine.

Recent Advances in the Roles of Neutrophils in Toxoplasmosis.

Neutrophils are now recognized as major components of the response to Toxoplasma gondii by their contribution to parasite elimination by a number of mechanisms. This article focuses on recent advances in the understanding of the mechanisms of migration, cytokine release, and formation of extracellular traps by neutrophils during toxoplasmosis.

Babesia divergens glycosylphosphatidylinositols modulate blood coagulation and induce Th2-biased cytokine profiles in antigen presenting cells.

Glycosylphosphatidylinositols (GPIs) are glycolipids described as toxins of protozoan parasites due to their inflammatory properties in mammalian hosts characterized by the production of interleukin (IL)-1, IL-12 and tumor necrosis factor (TNF)-α. In the present work, we studied the cytokines produced by antigen presenting cells in response to ten different GPI species extracted from Babesia divergens, responsible for babesiosis. Interestingly, B. divergens GPIs induced the production of anti-inflammatory cytokines (IL-2, IL-5) and of the regulatory cytokine IL-10 by macrophages and dendritic cells. In contrast to all protozoan GPIs studied until now, GPIs from B. divergens did not stimulate the production of TNF-α and IL-12, leading to a unique Th1/Th2 profile. Analysis of the carbohydrate composition of the B. divergens GPIs indicated that the di-mannose structure was different from the evolutionary conserved tri-mannose structure, which might explain the particular cytokine profile they induce. Expression of major histocompatibility complex (MHC) molecules on dendritic cells and apoptosis of mouse peritoneal cells were also analysed. B. divergens GPIs did not change expression of MHC class I, but decreased expression of MHC class II at the cell surface, while GPIs slightly increased the percentages of apoptotic cells. During pathogenesis of babesiosis, the inflammation-coagulation auto-amplification loop can lead to thrombosis and the effect of GPIs on coagulation parameters was investigated. Incubation of B. divergens GPIs with rat plasma ex vivo led to increase of fibrinogen levels and to prolonged activated partial thromboplastin time, suggesting a direct modulation of the extrinsic coagulation pathway by GPIs.

In vitro cellular responses to Neospora caninum glycosylphosphatidylinositols depend on the host origin of antigen presenting cells.

Neosporosis due to Neospora caninum causes abortions in farm animals such as cattle. No treatment and vaccine exist to fight this disease, responsible for considerable economic losses. It is thus important to better understand the immune responses occurring during the pathogenesis to control them in a global strategy against the parasite. In this context, we studied the roles of N. caninum glycosylphosphatidylinositols (GPIs), glycolipids defined as toxins in the related parasite Plasmodium falciparum. We demonstrated for the first time that GPIs could be excreted in the supernatant of N. caninum culture and trigger cell signalling through the Toll-like receptors 2 and 4. In addition, antibodies specific to N. caninum GPIs were detected in the serum of infected mice. As shown for other protozoan diseases, they could play a role in neutralizing GPIs. N. caninum GPIs were able to induce the production of tumour necrosis factor-α, interleukin(IL)-1ß and IL-12 cytokines by murine macrophages and dendritic cells. Furthermore, GPIs significantly reduced expression of major histocompatibility complex (MHC) molecules of class I on murine dendritic cells. In contrast to murine cells, bovine blood mononuclear cells produced increased levels of IFN-γ and IL-10, but reduced levels of IL-12p40 in response to GPIs. On these bovine cells, GPI had the tendency to up-regulate MHC class I, but to down-regulate MHC class II. Altogether, these results suggest that N. caninum GPIs might differentially participate in the responses of antigen presenting cells induced by the whole parasite in mouse models of neosporosis and in the natural cattle host.

Imidazo[1,2-b]pyridazines targeting Toxoplasma gondii calcium-dependent protein kinase 1 decrease the parasite burden in mice with acute toxoplasmosis.

The current therapeutic arsenal for toxoplasmosis is restricted to drugs non-specific to the parasite which cause important side effects. Development of more efficient and specific anti-Toxoplasma compounds is urgently needed. Imidazo[1,2-b]pyridazines designed to inhibit the calcium-dependent protein kinase 1 of Toxoplasma gondii (TgCDPK1) and effective against tachyzoite growth in vitro at submicromolar ranges were modified into hydrochloride salts to be administered in vivo in a mouse model of acute toxoplasmosis. All protonated imidazo[1,2-b]pyridazine salts (SP230, SP231 and SP232) maintained their activity on TgCDPK1 and T. gondii tachyzoites. Rat and mouse liver microsomes were used to predict half-life and intrinsic clearance, and the pharmacokinetic profile of the most rapidly degraded imidazo[1,2b]pyridazine salt (SP230) was determined in serum, brain and lungs of mice after a single administration of 50 mg/kg. Compounds were then tested in vivo in a murine model of acute toxoplasmosis. Mice infected with tachyzoites of the ME49 strain of T. gondii were treated for 4, 7 or 8 days with 25 or 50 mg/kg/day of SP230, SP231 or SP232. The parasite burdens were strongly diminished (>90% reduction under some conditions) in the spleen and the lungs of mice treated with imidazo[1,2-b]pyridazine salts compared with untreated mice, without the need for pre-treatment. Moreover, no increases in the levels of hepatic and renal toxicity markers were observed, demonstrating no significant signs of short-term toxicity. To conclude, imidazo[1,2-b]pyridazine salts have strong efficacy in vivo on acute toxoplasmosis and should be further tested in a model of mouse congenital toxoplasmosis.

Extracts of Tectona grandis and Vernonia amygdalina have anti-Toxoplasma and pro-inflammatory properties in vitro.

Tectona grandis (teak) and Vernonia amygdalina (bitter leaf) are plants used in traditional medicine in West Africa. In this study, we tested ethanolic and hydro-ethanolic extracts of bark and leaves of T. grandis and ethanolic extract of leaves of V. amygdalina for their inhibitory effect on Toxoplasma gondii, a protozoan parasite responsible for toxoplasmosis. Ethanolic extract of V. amygdalina leaves had proportional contents of phenols, tannins, flavonoids, and polysaccharides. This extract presented the highest efficacy against T. gondii, the lowest cytotoxicity to mammalian cells, but moderate anti-oxidant activity compared to other plant extracts. Ethanolic extract of T. grandis bark also had elevated anti-T. gondii activity, low cytotoxicity on mammalian cells, and one of the highest anti-oxidant activities. However, the phytochemical content of this extract was not very different from the hydro-ethanolic extract, which had no anti-T. gondii activity. In addition, ethanolic extract of V. amygdalina leaves, but not of T. grandis bark, significantly increased the production of TNF-α and NO by antigen-presenting cells. Both extracts had the tendency to decrease expression of major histocompatibility complex molecules at the surface of antigen-presenting cells, while they did not modulate the percentage of apoptotic cells. A study of signalling pathways would help to determine the mechanisms of action of these plant extracts.

TITLE: Les extraits de Tectona grandis et de Vernonia amygdalina ont des propriétés anti-Toxoplasma et pro-inflammatoires in vitro. ABSTRACT: Tectona grandis (teck) et Vernonia amygdalina sont des plantes utilisées dans la médecine traditionnelle en Afrique de l'Ouest. Dans cette étude, l'effet inhibiteur d'extraits éthanoliques et hydro-éthanoliques d'écorce et de feuilles de T. grandis et de l'extrait éthanolique des feuilles de V. amygdalina a été étudié sur Toxoplasma gondii, un parasite protozoaire responsable de la toxoplasmose. L'extrait éthanolique des feuilles de V. amygdalina avait des quantités équivalentes de phénols, tanins, flavonoïdes et polysaccharides. Cet extrait présentait la plus grande efficacité contre T. gondii, la plus faible cytotoxicité vis-à-vis de cellules de mammifères, mais une activité anti-oxydante moyenne comparée aux autres extraits de plantes. L'extrait éthanolique d'écorce de T. grandis avait aussi une activité anti-T. gondii élevée, une faible cytotoxicité vis-à-vis des cellules de mammifères et l'une des activités anti-oxydantes les plus élevées. Cependant, le contenu phytochimique de cet extrait n'était pas très différent de l'extrait hydro-éthanolique qui n'avait pas d'activité anti-T. gondii. De plus, l'extrait éthanolique des feuilles de V. amygdalina, mais pas de l'écorce de T. grandis, augmentait significativement la production de TNF-α et de NO par les cellules présentatrices d'antigènes. Les deux extraits avaient tendance à diminuer l'expression des molécules du complexe majeur d'histocompatibilité à la surface des cellules présentatrices d'antigènes alors qu'ils ne modulaient pas le pourcentage de cellules apoptotiques. L'étude des voies de signalisation permettrait de comprendre les mécanismes d'action de ces extraits de plantes.

Development of new highly potent imidazo[1,2-b]pyridazines targeting Toxoplasma gondii calcium-dependent protein kinase 1.

Using a structure-based design approach, we have developed a new series of imidazo[1,2-b]pyridazines, targeting the calcium-dependent protein kinase-1 (CDPK1) from Toxoplasma gondii. Twenty derivatives were thus synthesized. Structure-activity relationships and docking studies confirmed the binding mode of these inhibitors within the ATP binding pocket of TgCDPK1. Two lead compounds (16a and 16f) were then identified, which were able to block TgCDPK1 enzymatic activity at low nanomolar concentrations, with a good selectivity profile against a panel of mammalian kinases. The potential of these inhibitors was confirmed in vitro on T. gondii growth, with EC50 values of 100 nM and 70 nM, respectively. These best candidates also displayed low toxicity to mammalian cells and were selected for further in vivo investigations on murine model of acute toxoplasmosis.

A small-molecule cell-based screen led to the identification of biphenylimidazoazines with highly potent and broad-spectrum anti-apicomplexan activity.

An in vitro screening of the anti-apicomplexan activity of 51 compounds, stemming from our chemical library and from chemical synthesis, was performed. As a study model, we used Toxoplasma gondii (T. gondii), expressing ß-galactosidase for the colorimetric assessment of drug activity on parasites cultivated in vitro. This approach allowed the validation of a new series of molecules with a biphenylimidazoazine scaffold as inhibitors of T. gondii growth in vitro. Hence, 8 molecules significantly inhibited intracellular replication of T. gondii in vitro, with EC50 < 1 µM, while being non-toxic for human fibroblasts at these concentrations. Most attractive candidates were then selected for further biological investigations on other apicomplexan parasites (Neospora caninum, Besnoitia besnoiti, Eimeria tenella and Plasmodium falciparum). Finally, two compounds were able to inhibit growth of four different apicomplexans with EC50 in the submicromolar to nanomolar range, for each parasite. These data, including the broad anti-parasite spectrum of these inhibitors, define a new generation of potential anti-parasite compounds of wide interest, including for veterinary application. Studies realized on E. tenella suggest that these molecules act during the intracellular development steps of the parasite. Further experiments should be done to identify the molecular target(s) of these compounds.

Virulent and avirulent strains of Toxoplasma gondii which differ in their glycosylphosphatidylinositol content induce similar biological functions in macrophages.

Glycosylphosphatidylinositols (GPIs) from several protozoan parasites are thought to elicit a detrimental stimulation of the host innate immune system aside their main function to anchor surface proteins. Here we analyzed the GPI biosynthesis of an avirulent Toxoplasma gondii type 2 strain (PTG) by metabolic radioactive labeling. We determined the biological function of individual GPI species in the PTG strain in comparison with previously characterized GPI-anchors of a virulent strain (RH). The GPI intermediates of both strains were structurally similar, however the abundance of two of six GPI intermediates was significantly reduced in the PTG strain. The side-by-side comparison of GPI-anchor content revealed that the PTG strain had only ∼ 34% of the protein-free GPIs as well as ∼ 70% of the GPI-anchored proteins with significantly lower rates of protein N-glycosylation compared to the RH strain. All mature GPIs from both strains induced comparable secretion levels of TNF-α and IL-12p40, and initiated TLR4/MyD88-dependent NF-κBp65 activation in macrophages. Taken together, these results demonstrate that PTG and RH strains differ in their GPI biosynthesis and possess significantly different GPI-anchor content, while individual GPI species of both strains induce similar biological functions in macrophages.

Trypanosoma cruzi parasites fight for control of the JAK-STAT pathway by disarming their host.

The zoonotic Chagas' disease is caused by infections with the hemoflagellate Trypanosoma cruzi (T. cruzi) which is endemic in Latin America. Despite recent advances in our understanding of the pathogenesis of the disease, the underlying molecular processes involved in host-parasite interactions are only poorly understood. In particular, the mechanisms for parasite persistence in host cells remain largely unknown. Cytokine-driven transcription factors from the family of STAT (signal transducer and activator of transcription) proteins appear to play a central role in the fight against T. cruzi infection. However, amastigotes proliferating in the cytoplasm of infected host cells develop effective strategies to circumvent the attack executed by STAT proteins. This review highlights the interactions between T. cruzi parasites and human host cells in terms of cytokine signaling and, in particular, discusses the impact of STATs on the balance between parasite invasion and clearance.

Trypomastigotes and amastigotes of Trypanosoma cruzi induce apoptosis and STAT3 activation in cardiomyocytes in vitro.

The haemoflagellate Trypanosoma cruzi is the causative agent of Chagas' disease that occurs in approximately 8 million people in Latin America. Patients infected with T. cruzi frequently suffer of cardiomegaly and may die of myocardial failure. Here we show that T. cruzi trypomastigotes (extracellular form) increased in vitro apoptosis of rat cardiomyocytes. Additionally, we demonstrated that amastigotes (intracellular form), for which a method for purification was established, were also able to induce cardiomyocyte apoptosis. Increase of apoptosis was associated with up-regulation of the apoptotic gene bax by trypomastigotes, while expression of the anti-apoptotic gene bcl-2 was down-regulated by amastigotes. The transcription factor STAT3 but not STAT1 was activated in cardiomyocytes by trypomastigotes. In addition, tlr7 gene expression was up-regulated in cardiomyocytes incubated with trypomastigotes, suggesting that this Toll-like receptor is involved in the intracellular recognition after host cell invasion by T. cruzi. Glycosylphosphatidylinositols purified from trypomastigotes did not induce cardiomyocyte apoptosis and STAT activation but down-regulated tlr7 gene expression. In conclusion, cardiomyopathy observed in Chagas' disease might be in part due to apoptosis of cardiomyocytes induced directly by the parasite.

Sequencing of the smallest Apicomplexan genome from the human pathogen Babesia microti.

We have sequenced the genome of the emerging human pathogen Babesia microti and compared it with that of other protozoa. B. microti has the smallest nuclear genome among all Apicomplexan parasites sequenced to date with three chromosomes encoding ∼3500 polypeptides, several of which are species specific. Genome-wide phylogenetic analyses indicate that B. microti is significantly distant from all species of Babesidae and Theileridae and defines a new clade in the phylum Apicomplexa. Furthermore, unlike all other Apicomplexa, its mitochondrial genome is circular. Genome-scale reconstruction of functional networks revealed that B. microti has the minimal metabolic requirement for intraerythrocytic protozoan parasitism. B. microti multigene families differ from those of other protozoa in both the copy number and organization. Two lateral transfer events with significant metabolic implications occurred during the evolution of this parasite. The genomic sequencing of B. microti identified several targets suitable for the development of diagnostic assays and novel therapies for human babesiosis.

Defibrotide interferes with several steps of the coagulation-inflammation cycle and exhibits therapeutic potential to treat severe malaria.

OBJECTIVE: The coagulation-inflammation cycle has been implicated as a critical component in malaria pathogenesis. Defibrotide (DF), a mixture of DNA aptamers, displays anticoagulant, anti-inflammatory, and endothelial cell (EC)-protective activities and has been successfully used to treat comatose children with veno-occlusive disease. DF was investigated here as a drug to treat cerebral malaria. METHODS AND RESULTS: DF blocks tissue factor expression by ECs incubated with parasitized red blood cells and attenuates prothrombinase activity, platelet aggregation, and complement activation. In contrast, it does not affect nitric oxide bioavailability. We also demonstrated that Plasmodium falciparum glycosylphosphatidylinositol (Pf-GPI) induces tissue factor expression in ECs and cytokine production by dendritic cells. Notably, dendritic cells, known to modulate coagulation and inflammation systemically, were identified as a novel target for DF. Accordingly, DF inhibits Toll-like receptor ligand-dependent dendritic cells activation by a mechanism that is blocked by adenosine receptor antagonist (8-p-sulfophenyltheophylline) but not reproduced by synthetic poly-A, -C, -T, and -G. These results imply that aptameric sequences and adenosine receptor mediate dendritic cells responses to the drug. DF also prevents rosetting formation, red blood cells invasion by P. falciparum and abolishes oocysts development in Anopheles gambiae. In a murine model of cerebral malaria, DF affected parasitemia, decreased IFN-γ levels, and ameliorated clinical score (day 5) with a trend for increased survival. CONCLUSION: Therapeutic use of DF in malaria is proposed.

Glycosylphosphatidylinositols of Toxoplasma gondii induce matrix metalloproteinase-9 production and degradation of galectin-3.

Toxoplasma gondii glycosylphosphatidylinositols (GPIs) bind to galectin-3 to induce TNF-α production in macrophages via Toll-like receptors 2 and 4. Here we show that T. gondii GPIs stimulate human macrophages to synthesize matrix metalloproteinase-9 in a TNF-α-dependent pathway and degrade extracellular galectin-3.

Immunological responses induced by a DNA vaccine expressing RON4 and by immunogenic recombinant protein RON4 failed to protect mice against chronic toxoplasmosis.

The development of an effective vaccine against Toxoplasma gondii infection is an important issue due to the seriousness of the related public health problems, and the economic importance of this parasitic disease worldwide. Rhoptry neck proteins (RONs) are components of the moving junction macromolecular complex formed during invasion. The aim of this study was to evaluate the vaccine potential of RON4 using two vaccination strategies: DNA vaccination by the intramuscular route, and recombinant protein vaccination by the nasal route. We produced recombinant RON4 protein (RON4S2) using the Schneider insect cells expression system, and validated its antigenicity and immunogenicity. We also constructed optimized plasmids encoding full length RON4 (pRON4), or only the N-terminal (pNRON4), or the C-terminal part (pCRON4) of RON4. CBA/J mice immunized with pRON4, pNRON4 or pCRON4 plus a plasmid encoding the granulocyte-macrophage-colony-stimulating factor showed high IgG titers against rRON4S2. Mice immunized by the nasal route with rRON4S2 plus cholera toxin exhibited low levels of anti-RON4S2 IgG antibodies, and no intestinal IgA antibodies specific to RON4 were detected. Both DNA and protein vaccination generated a mixed Th1/Th2 response polarized towards the IgG1 antibody isotype. Both DNA and protein vaccination primed CD4+ T cells in vivo. In addition to the production of IFN-γ, and IL-2, Il-10 and IL-5 were also produced by the spleen cells of the immunized mice stimulated with RON4S2, suggesting that a mixed Th1/Th2 type immune response occurred in all the immunized groups. No cytokine was detectable in stimulated mesenteric lymph nodes from mice immunized by the nasal route. Immune responses were induced by both DNA and protein vaccination, but failed to protect the mice against a subsequent oral challenge with T. gondii cysts. In conclusion, strategies designed to enhance the immunogenicity and to redirect the cellular response towards a Th1 type response against RON4 could lead to more encouraging results.