Faculty Bibliography

Malaria, leishmaniasis and trypanosomiasis are arthropod-borne, parasitic diseases that constitute a major global health problem. They are generally found in developing countries, where lack of access to preventive tools and treatment hinders their management. Because these parasites share an increased demand on glucose consumption with most cancer cells, six compounds used in anti-tumoral research were selected to be tested as antiparasitic agents in in vitro models of Leishmania infantum, Trypanosoma brucei, T. cruzi, and Plasmodium falciparum: dichloroacetic acid (DCA), 3-bromopyruvic acid (3BP), 2-deoxy-D-glucose (2DG), lonidamine (LND), metformin (MET), and sirolimus (SIR). No parasite-killing activity was found in L. infantum promastigotes, whereas DCA and 3BP reduced the burden of intra-macrophagic amastigotes. For T. brucei all selected compounds, but 2DG, decreased parasite survival. DCA, 2DG, LND and MET showed parasite-killing activity in T. cruzi. Finally, anti-plasmodial activity was found for DCA, 2DG, LND, MET and SIR. These results reinforce the hypothesis that drugs with proven efficacy in the treatment of cancer by interfering with ATP production, proliferation, and survival cell strategies might be useful in treating threatening parasitic diseases and provide new opportunities for their repurposing.

Malaria caused by Plasmodium vivax is a highly prevalent infection world-wide, that was previously considered mild, but complications such as anemia have been highly reported in the past years. In mice models of malaria, anti-phosphatidylserine (anti-PS) autoantibodies, produced by atypical B-cells, bind to uninfected erythrocytes and contribute to anemia. In human patients with P. falciparum malaria, the levels of anti-PS, atypical B-cells and anemia are strongly correlated to each other. In this study, we focused on assessing the relationship between autoantibodies, different B-cell populations and hemoglobin levels in two different cohorts of P. vivax patients from Colombia, South America. In a first longitudinal cohort, our results show a strong inverse correlation between different IgG autoantibodies tested (anti-PS, anti-DNA and anti-erythrocyte) and atypical memory B-cells (atMBCs) with hemoglobin in both P. vivax and P. falciparum patients over time. In a second cross-sectional cohort, we observed a stronger relation between hemoglobin levels, atMBCs and autoantibodies in complicated P. vivax patients compared to uncomplicated ones. Altogether, these data constitute the first evidence of autoimmunity associating with anemia and complicated P. vivax infections, suggesting a role for its etiology through the expansion of autoantibody-secreting atMBCs.

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi. It is endemic in South and Central America and recently has been found in other parts of the world, due to migration of chronically infected patients. The current treatment for Chagas disease is not satisfactory, and there is a need for new treatments. In this work, we describe the optimization of a hit compound resulting from the phenotypic screen of a library of compounds against T. cruzi. The compound series was optimized to the level where it had satisfactory pharmacokinetics to allow an efficacy study in a mouse model of Chagas disease. We were able to demonstrate efficacy in this model, although further work is required to improve the potency and selectivity of this series.

Utilizing a target repurposing and parasite-hopping approach, we tested a previously reported library of compounds that were active against Trypanosoma brucei, plus 31 new compounds, against a variety of protozoan parasites including Trypanosoma cruzi, Leishmania major, Leishmania donovani, and Plasmodium falciparum. This led to the discovery of several compounds with submicromolar activities and improved physicochemical properties that are early leads toward the development of chemotherapeutic agents against kinetoplastid diseases and malaria.

Trypanosoma brucei (T. brucei) and Trypanosoma cruzi (T. cruzi) are causative agents of parasitic diseases known as human African trypanosomiasis and Chagas disease, respectively. Together, these diseases affect 68 million people around the world. Current treatments are unsatisfactory, frequently associated with intolerable side-effects, and generally inadequate in treating all stages of disease. In this paper, we report the discovery of N-ethylurea pyrazoles that potently and selectively inhibit the viability of T. brucei and T. cruzi. Sharp and logical SAR led to the identification of 54 as the best compound, with an in vitro IC₅₀ of 9 nM and 16 nM against T. b. brucei and T. cruzi, respectively. Compound 54 demonstrates favorable physicochemical properties and was efficacious in a murine model of Chagas disease, leading to undetectable parasitemia within 6 days when CYP metabolism was inhibited.

Severe anemia is a major cause of death by malaria. The loss of uninfected erythrocytes is an important contributor to malarial anemia; however, the mechanisms underlying this pathology are not well understood. Malaria-induced anemia is related to autoimmune antibodies against the membrane lipid phosphatidylserine (PS). In mice, these antibodies induce the clearance of uninfected erythrocytes after binding to PS exposed in their membrane. In human malaria patients there is a strong correlation between anemia and anti-PS antibodies. During malaria, anti-PS antibodies are produced by atypical B cells, whose levels correlate with the development of anemia in patients. Autoimmune responses, which are documented frequently in different infections, contribute to the pathogenesis of malaria by inducing the clearance of uninfected erythrocytes.

BACKGROUND:Tamoxifen is an oestrogen receptor modulator that is widely used for the treatment of early stage breast cancer and reduction of recurrences. Tamoxifen is also used as a powerful research tool for controlling gene expression in the context of the Cre/loxP site-specific recombination system in conditional mutant mice. METHODS:To determine whether the administration of tamoxifen affects Plasmodium growth and/or disease outcome in malaria, in vitro studies assessing the effect of tamoxifen and its active metabolite 4-hydroxytamoxifen on Plasmodium falciparum blood stages were performed. Tamoxifen effects were also evaluated in vivo treating C57/B6 mice infected with Plasmodium berghei (ANKA strain), which is the standard animal model for the study of cerebral malaria. RESULTS:Tamoxifen and its active metabolite, 4-hydroxytamoxifen, show activity in vitro against P. falciparum (16.7 to 5.8 µM IC50, respectively). This activity was also confirmed in tamoxifen-treated mice infected with P. berghei, which show lower levels of parasitaemia and do not develop signs of cerebral malaria, compared to control mice. Mice treated with tamoxifen for 1 week and left untreated for an additional week before infection showed similar parasitaemia levels and signs of cerebral malaria as control untreated mice. CONCLUSIONS:Tamoxifen and its active metabolite, 4-hydroxytamoxifen, have significant activity against the human parasite P. falciparum in vitro and the rodent parasite P. berghei in vivo. This activity may be useful for prevention of malaria in patients taking this drug chronically, but also represents a major problem for scientists using the conditional mutagenic Cre/LoxP system in the setting of rodent malaria. Allowing mice to clear tamoxifen before starting a Plasmodium infection allows the use the Cre/LoxP conditional mutagenic system to investigate gene function in specific tissues.

Anemia is a common complication of malaria that is characterized by the loss of infected and uninfected erythrocytes. In mouse malaria models, clearance of uninfected erythrocytes is promoted by autoimmune anti-phosphatidylserine (PS) antibodies produced by T-bet⁺B-cells, which bind to exposed PS in erythrocytes, but the mechanism in patients is still unclear. In Plasmodium falciparum patients with anemia, we show that atypical memory FcRL5⁺T-bet⁺ B-cells are expanded and associate both with higher levels of anti-PS antibodies in plasma and with the development of anemia in these patients. No association of anti-PS antibodies or anemia with other B-cell subsets and no association of other antibody specificities with FcRL5⁺T-bet⁺ B-cells is observed, revealing high specificity in this response. We also identify FcRL5⁺T-bet⁺ B-cells as producers of anti-PS antibodies in ex vivo cultures of naïve human peripheral blood mononuclear cells (PBMC) stimulated with P.-falciparum-infected erythrocyte lysates. These data define a crucial role for atypical memory B-cells and anti-PS autoantibodies in human malarial anemia.

Malaria is a highly inflammatory disease caused by the protozoan parasite Plasmodium During the blood stage of infection, patients exhibit fever with high levels of inflammatory cytokines in their blood. However, when cells of the immune system are incubated with the parasite in vitro, their cytokine response is low. In particular, human primary dendritic cells (DCs) respond to Plasmodium falciparum-infected erythrocytes by upregulating maturation markers and chemokines but lack a substantial cytokine response. Because oxidative stress is a trigger of inflammatory cytokines in malaria and synergizes with P. falciparum to induce IL-1β secretion by macrophages, we assessed whether oxidative stress has an impact on DC maturation and function in response to P. falciparum Using xanthine oxidase, a reactive oxygen species- (ROS) producing enzyme that is increased during malaria, we observed that exposure to extracellular ROS potentiated DC maturation in response to the parasite. Xanthine oxidase-derived ROS increased parasite-induced cytokine secretion and CD80 surface expression in DCs. This enhanced maturation phenotype boosted the DCs' ability to prime autologous naive CD4⁺ T cells, resulting in higher T cell proliferation in vitro. Xanthine oxidase-derived ROS did not have an effect on the cytokines produced by primed T cells. We propose that oxidative stress during malaria contributes to the inflammatory response by enhancing the magnitude of DC and CD4⁺ T cell responses without changing the quality.