Chagasi Parasites Research Articles

In Vitro Activity Against Trypanosoma cruzi and Leishmania chagasi Parasites of 2,4-Diaryl 1,2,3,4-Tetrahydroquinoline Derivatives

Diverse polyfunctionalized tetrahydroquinolines, easily prepared using BF3 .OEt2-catalyzed three component imino Diels–Alder reaction, were tested in vitro against Trypanosoma cruzi and Leishmania chagasi parasites and also for cytotoxicity using Vero and THP-1 mammalian cell lines. These studies showed that the most active compound was 7h (IC50 5.77 ± 0.21; IC90 40.43 ± 1.58 μM for T. cruzi and IC50 0.27 ± 0.03; IC90 2.16± 0.37 μM for L. chagasi) has selective index (SI) values greater than the reference drugs (SI > 46 and SI > 1000 in epimastigotes form of T. cruzi and promastigotes form of L. chagasi parasites, respectively). Moreover, the comp. 7h did not show cytotoxicity against Vero or THP-1 cells (IC50 > 270 μM). Keywords: Antiparasitic activities, Cytotoxicity, Imino Diels-Alder reaction, Leishmania chagasi, Tetrahydroquinolines, Trypanosoma cruzi, paromomycin, Leishmania and Trypanosoma, flagellated protozoa, anti-leishmanial

Leishmania infection in humans, dogs and sandflies in a visceral leishmaniasis endemic area in Maranhão, Brazil

Leishmania infection in humans, dogs and sandflies was examined in the endemic visceral leishmaniasis (VL) municipality of Raposa, state of Maranhão, Brazil. In this study, we examined Leishmania chagasi infection in the blood serum of both humans and Canis familiaris and the natural Leishmania sp. infection rate in the sandfly vector, Lutzomyia longipalpis. Enzyme-linked immunosorbent assay, indirect immunofluorescence reaction and polymerase chain reaction were performed to detect Leishmania infections in humans, dogs and sandflies, respectively. Overall, 186 out of 986 studied human beings were infected with L. chagasi parasites, representing an infection prevalence of 18.9%. An even higher infection rate was detected in dogs, where 66 (47.8%) out of 138 were infected. Among all Lu. longipalpis captured (n = 1,881), only 26.7% were females. The Leishmania infection frequency for the vector Lu. longipalpis was 1.56%. Remarkably, all infected sandflies were found in the peridomiciliary area. Furthermore, a high incidence of asymptomatic forms of VL in the human and canine populations was observed. The results of this study suggest autochthonous transmission of L. chagasi in this endemic area for visceral leishmaniasis because infection by Leishmania sp. was identified in all important elements of the transmission chain.

In vitro and experimental therapeutic studies of the calcium channel blocker bepridil: Detection of viable Leishmania (L.) chagasi by real-time PCR

The need for novel and efficacious drugs against neglected parasitic diseases, such as Leishmaniasis and American Trypanosomiasis, is certainly apparent. In this work, we evaluated the in vitro potential of the calcium channel blocker bepridil against Leishmania spp. and Trypanosoma cruzi parasites and exploited an experimental assay using a hamster model with Leishmania (L.) chagasi, with a real-time PCR method for therapeutic evaluation. Bepridil was in vitro effective against promastigotes and intracellular amastigotes of L. (L.) chagasi, with 50% inhibitory concentration (IC50) values of 3.81 and 21.55μM, respectively. Leishmania (L.) amazonensis, L. (L.) major and L. (V.) braziliensis promastigotes and T. cruzi trypomastigotes were also susceptible to bepridil, with in vitro selectivity toward parasites and IC50 values in the range of 3 to 7μM. The mammalian cytotoxicity using LLC-MK2 cells resulted in an IC50 value of 62.67μM. However, bepridil showed lack of activity at 12mg/kg in the experimental hamster model infected with L. (L.) chagasi parasites. However, the real-time PCR was a promising tool for the accurate and fast quantification of RNA of living parasites in the liver and spleen of infected hamsters after treatment, eliminating time-consuming light microscopy evaluations.

Reduced Hamster Usage and Stress in Propagating Leishmania chagasi Promastigotes Using Cryopreservation and Saphenous Vein Inoculation

Leishmania chagasi, a causal agent of visceral leishmaniasis, requires passage through lab animals such as hamsters to maintain its virulence. Hamster infection is typically accomplished via cardiac puncture or intraperitoneal injection, procedures accompanied by risks of increased animal stress and death. The use of the hamster model also necessitates a regular supply of infected animals, because L. chagasi parasites newly isolated from an infected hamster can be grown in culture for only several weeks before loss of function/phenotype occurs. In an effort to decrease animal usage and animal stress, experiments were performed to assess a more gentle inoculation procedure (saphenous vein inoculation) and the use of cryopreserved parasite cells for research experiments. Of 81 hamsters inoculated by the saphenous vein, 80 became infected as determined ante mortem, by display of clinical symptoms of leishmaniasis (onset of symptoms at 105 +/- 22 days post-inoculation), and postmortem by the presence of parasites within the spleen. Splenic parasite load calculated for a subset (n = 34) of infected hamsters was 124 to 26,177 Leishmania donovani infection units. Cryopreserved, and never-stored, cells were equivalent in all properties evaluated, including developmental changes in morphology during culture, culture growth rates, parasite resistance to serum-mediated lysis, and expression of developmentally regulated surface proteins major surface protease and promastigote surface antigen.

Macrophage and T-Cell Gene Expression in a Model of Early Infection with the Protozoan Leishmania chagasi

Visceral leishmaniasis is a potentially fatal infectious disease caused by the protozoan parasite Leishmania infantum/chagasi in the New World, or by L. donovani or L. infantum/chagasi in the Old World. Infection leads to a variety of outcomes ranging from asymptomatic infection to active disease, characterized by fevers, cachexia, hepatosplenomegaly and suppressed immune responses. We reasoned that events occurring during the initial few hours when the parasite encounters cells of the innate and adaptive immune systems are likely to influence the eventual immune response that develops. Therefore, we performed gene expression analysis using Affymetrix U133Plus2 microarray chips to investigate a model of early infection with human monocyte-derived macrophages (MDMs) challenged with wild-type L. chagasi parasites, with or without subsequent co-culture with Leishmania-naïve, autologous T-cells. Microarray data generated from total RNA were analyzed with software from the Bioconductor Project and functional clustering and pathway analysis were performed with DAVID and Gene Set Enrichment Analysis (GSEA), respectively. Many transcripts were down-regulated by infection in cultures containing macrophages alone, and the pattern indicated a lack of a classically activated phenotype. By contrast, the addition of autologous Leishmania-naïve T cells to infected macrophages resulted in a pattern of gene expression including many markers of type 1 immune cytokine activation (IFN-γ, IL-6, IL-1α, IL-1β). There was simultaneous up-regulation of a few markers of immune modulation (IL-10 cytokine accumulation; TGF-β Signaling Pathway). We suggest that the initial encounter between L. chagasi and cells of the innate and adaptive immune system stimulates primarily type 1 immune cytokine responses, despite a lack of classical macrophage activation. This local microenvironment at the site of parasite inoculation may determine the initial course of immune T-cell development.

Synthesis and in vitro activity of new tetrahydronaphtho[1,2- b]azepine derivatives against Trypanosoma cruzi and Leishmania chagasi parasites

Series of 2- exo-aryl-1,4-epoxy-2,3,4,5-tetrahydronaphtho[1,2- b]azepines 3a– k and cis-2-aryl-4-hydroxy-2,3,4,5-tetrahydronaphtho[1,2- b]azepines 4a– j were synthesized and evaluated against free and intracellular live forms of Trypanosoma cruzi and Leishmania chagasi parasites using in vitro assays. Cell toxicity was also analyzed on Vero and THP-1 mammalian cell lines. The compounds 3c, 3f, and 4d were the most active against both live forms of T. cruzi parasites with low mammalian cell toxicity. Some compounds were active on free live forms of L. chagasi parasites but none was active on intracellular amastigotes of L. chagasi infecting THP-1 macrophages.

Target-Oriented Synthesis of Antiparasitic 2-Hetaryl Substituted Quinolines Based on Imino Diels-Alder Reactions

Target-oriented synthesis of new substituted 2-hetarylquinolines 6-19 and their in vitro activity against endemic parasites such as Leishmania chagasi and Trypanosoma cruzi are reported. In addition, their cytotoxicities for mammalian Vero cells and THP-1 macrophages cells are assessed. These studies showed that between the new (tetrahydro)quinolines series tested in these assays, the 4-methyl-8-isopropyl-2-(3- pyridinyl)quinoline 15 exhibits the best in vitro response against both live forms of T. cruzi and L. chagasi parasites and displays no cytotoxic activity on mammalian cells.

Atypical cutaneous leishmaniasis cases display elevated antigen‐induced interleukin‐10

In humans, Leishmania chagasi parasites can produce subclinical infections, atypical cutaneous leishmaniasis (ACL) and visceral leshmaniasis that is potentially fatal if not treated in a timely fashion. L. chagasi parasites that cause both ACL and visceral disease appear to be genetically similar, which suggests that host factors such as the immune response play an important role in controlling infection. We evaluated the immunologic response in ACL using peripheral blood mononuclear cells (PBMCs) of 37 subjects divided into three groups: (i) active ACL cases, (ii) asymptomatic cases and (iii) persons with no history of Leishmania infection. The supernatants of stimulated PBMCs were analysed for production of IL-10, IFN-gamma and IL-2. Robust production of IL-10 in response to Leishmania stimulation was observed in active ACL cases, compared to low levels in asymptomatic cases and negative controls. Serum IgE levels, measured by ELISA, were not significantly different among the three groups. In addition, ACL cases displayed depressed levels of all cytokines in response to mitogen. Thus, this first characterization of the immune response in ACL suggests a role for IL-10 as well as partial immunosuppression.

A sensitive flow cytometric methodology for studying the binding of L. chagasito canine peritoneal macrophages

BackgroundThe Leishmania promastigote-macrophage interaction occurs through the association of multiple receptors on the biological membrane surfaces. The success of the parasite infection is dramatically dependent on this early interaction in the vertebrate host, which permits or not the development of the disease. In this study we propose a novel methodology using flow cytometry to study this interaction, and compare it with a previously described "in vitro" binding assay.MethodsTo study parasite-macrophage interaction, peritoneal macrophages were obtained from 4 dogs and adjusted to 3 × 106 cells/mL. Leishmania (Leishmania) chagasi parasites (stationary-phase) were adjusted to 5 × 107 cells/mL. The interaction between CFSE-stained Leishmania chagasi and canine peritoneal macrophages was performed in polypropylene tubes to avoid macrophage adhesion. We carried out assays in the presence or absence of normal serum or in the presence of a final concentration of 5% of C5 deficient (serum from AKR/J mice) mouse serum. Then, the number of infected macrophages was counted in an optical microscope, as well as by flow citometry. Macrophages obtained were stained with anti-CR3 (CD11b/CD18) antibodies and analyzed by flow citometry.ResultsOur results have shown that the interaction between Leishmania and macrophages can be measured by flow cytometry using the fluorescent dye CFSE to identify the Leishmania, and measuring simultaneously the expression of an important integrin involved in this interaction: the CD11b/CD18 (CR3 or Mac-1) β2 integrin.ConclusionFlow cytometry offers rapid, reliable and sensitive measurements of single cell interactions with Leishmania in unstained or phenotypically defined cell populations following staining with one or more fluorochromes.

Role of Peroxidoxins in Leishmania chagasiSurvival: EVIDENCE OF AN ENZYMATIC DEFENSE AGAINST NITROSATIVE STRESS

The mechanisms by which Leishmania parasites survive exposure to highly reactive oxygen (ROS) and nitrogen (RNS) species within phagosomes of macrophages are not well known. Recently it has been shown that RNS alone is sufficient and necessary to control Leishmania donovani infection in mice (Murray, H. W., and Nathan, C. F. (1999) J. Exp. Med. 189, 741-746). No enzymatic defense against RNS has been discovered in Leishmania to date. We have previously isolated two peroxidoxins (LcPxn1 and LcPxn2) from Leishmania chagasi and showed that recombinant LcPxn1 protein was capable of detoxifying hydrogen peroxide, hydroperoxide, and hydroxyl radicals (Barr, S. D., and Gedamu, L. (2001) J. Biol. Chem. 276, 34279-34287). In further characterizing the physiological role of peroxidoxins in Leishmania survival, we show here that recombinant LcPxn1 protein can detoxify RNS in addition to ROS, whereas recombinant LcPxn2 protein can only detoxify hydrogen peroxide. LcPxn1 and LcPxn2 are localized to the cytoplasm, and overexpression of LcPxn1 in L. chagasi parasites enhanced survival when exposed to exogenous ROS and RNS and enhanced survival within U937 macrophage cells. Site-directed mutagenesis studies revealed that the conserved Cys-52 residue is essential for detoxifying hydrogen peroxide, t-butyl hydroperoxide, and hydroxyl radicals, whereas the conserved Cys-173 residue is essential for detoxifying t-butyl hydroperoxide and peroxynitrite. This is the first report of an enzymatic defense against RNS in Leishmania.

Cloning, characterization and overexpression of two iron superoxide dismutase cDNAs from Leishmania chagasi: role in pathogenesis ,

We have isolated and characterized two superoxide dismutase (SOD) cDNAs from a Leishmania chagasi promastigote cDNA library using degenerate primers derived from conserved amino acid residues of previously isolated manganese and iron SODs. Comparison of these two L. chagasi SOD deduced amino acid sequences with previously isolated MnSOD and FeSOD amino acid sequences revealed that they have higher homology to, and complete conservation of, invariant residues found in iron-containing SODs. Southern blot analysis showed that one gene, L.c.FeSODA, is a single copy gene, whereas the other gene, L.c.FeSODB, belongs to a multi-gene family. Transcript levels and enzyme activities of L.c.FeSODA and L.c.FeSODB show differential stage expression, with higher levels present in the amastigote stage of the parasite compared to the promastigote stage. Expression of the L.c.FeSODs in an E. coli SOD null strain protected the bacteria against free radical generating agents. Overexpression of these FeSODs in L. chagasi parasites also showed enhanced protection against the free radical generating agents, paraquat and nitroprusside. The cloning, characterization and overexpression of the L.c.FeSODA and L.c.FeSODB genes and proteins demonstrates the possible role of SODs in Leishmania pathogenesis.