Double Staining with Fluorescent Tracers to Determine Myeloid Cell Migration of Leishmania-infected Cells from Mouse Skin to Lymphatic Tissues by Flow Cytometry.

Immune cell trafficking in steady-state conditions and inflammatory cell recruitment into injured tissues is crucial for the surveillance of the immune system and the maintenance of body homeostasis. Tracking the cell journey from the infection site in the skin to lymphoid tissues has been challenging, and is typically determined using fluorescent cell tracers, antibodies, or photoconvertible models. Here, we describe the detailed method to track Leishmania-infected myeloid cells migrating from the skin to lymphatic tissues by multiparametric flow cytometry. These methods involve labeling of infective Leishmania donovani parasites with fluorescent cell tracers and phenotyping of myeloid cells with fluorescent antibodies, to determine the infection status of migratory myeloid cells. We also describe the detailed protocol to trace donor monocytes transferred intradermally into recipient mice in Leishmania donovani infection. These protocols can be adapted to study skin-lymphoid tissue migration of dendritic cells, inflammatory monocytes, neutrophils, and other phagocytic myeloid cells in response to vaccine antigens and infection. Key features • Cell-tracking of cell-trace-labeled parasites and monocytes from the skin to lymphatic tissues after transference into donor mice. • Identification of migratory cells labeled with fluorescent cell tracers and antibodies by flow cytometry. • Isolation, labeling, and transference of bone marrow monocytes from donor mice into the skin of recipient mice. • Description of a double-staining technique with fluorescent cell tracers to determine cell and parasite dissemination from the skin to lymphoid tissues.

Hematological and Clinical Features Associated with Initial Poor Treatment Outcomes in Visceral Leishmaniasis Patients with and without HIV Coinfection in Gondar, Northwest Ethiopia.

Ethiopia is among the countries with a high leishmaniasis burden. In this retrospective review, we aimed to determine hematological and clinical features associated with initial poor treatment outcomes of visceral leishmaniasis (VL) patients. The majority of VL cases in this study had leucopenia (94.3%), thrombocytopenia (87.1%), and anemia (85.9%). HIV coinfection was present in 7.0% (n = 23) of VL cases. At the center, VL patients without HIV coinfection were treated with sodium stibogluconate and paromomycin combination, whereas HIV coinfected cases were treated with AmBisome and miltefosine combination therapy. End-of-treatment cure rates among HIV-positive and HIV-negative visceral leishmaniasis cases, respectively, were 52.2% and 96.9%. Case fatality rates were 34.8% and 2.7% in HIV-positive and HIV-negative cases, respectively. Overall, non-survivors in this study were more likely to have HIV (55.0% vs. 4.1%, p < 0.001), sepsis (15.0% vs. 1.4%, p = 0.019), and dyspnea (40.0% vs. 2.7%, p < 0.001) at admission. In this regard, particular attention to the management of superimposed disease conditions at admission, including sepsis, HIV, and dyspnea, is needed to improve VL patients' treatment outcomes. The inadequacy of the current treatments, i.e., AmBisome and miltefosine combination therapy, for HIV coinfected visceral leishmaniasis patients requires further attention as it calls for new treatment modalities.

Development of an ex vivo lymph node explant model for identification of novel molecules active against Leishmania major.

Leishmaniasis is a vector-borne zoonotic infection affecting people in tropical and subtropical regions of the world. Current treatments for cutaneous leishmaniasis are difficult to administer, toxic, expensive, and limited in effectiveness and availability. Here we describe the development and application of a medium-throughput screening approach to identify new drug candidates for cutaneous leishmaniasis using an ex vivo lymph node explant culture (ELEC) derived from the draining lymph nodes of Leishmania major-infected mice. The ELEC supported intracellular amastigote proliferation and contained lymph node cell populations (and their secreted products) that enabled the testing of compounds within a system that mimicked the immunopathological environment of the infected host, which is known to profoundly influence parasite replication, killing, and drug efficacy. The activity of known antileishmanial drugs in the ELEC system was similar to the activity measured in peritoneal macrophages infected in vitro with L. major. Using the ELEC system, we screened a collection of 334 compounds, some of which we had demonstrated previously to be active against L. donovani, and identified 119 hits, 85% of which were confirmed to be active by determination of the 50% effective concentration (EC50). We found 24 compounds (7%) that had an in vitro therapeutic index (IVTI; 50% cytotoxic/effective concentration [CC50]/EC50) > 100; 19 of the compounds had an EC50 below 1 µM. According to PubChem searchs, 17 of those compounds had not previously been reported to be active against Leishmania. We expect that this novel method will help to accelerate discovery of new drug candidates for treatment of cutaneous leishmaniasis.

Congenital transmission of experimental leishmaniasis in a hamster model.

Little information is available on transplacental transmission of Leishmania spp. We determined the frequency and impact of congenital infection caused by Leishmania panamensis or L. donovani in experimentally infected hamsters. A polymerase chain reaction showed that congenital transmission occurred in 25.8% (24 of 93) of offspring born to L. panamensis-infected hamsters and 14.6% (11 of 75) offspring born to L. donovani-infected hamsters. Mortality during lactation was higher in offspring born to L. panamensis-infected hamsters and offspring born to L. donovani-infected hamsters than controls, and lymphoproliferation to Leishmania was more frequent in offspring born to L. panamensis-infected hamsters (17.4%, 11 of 63) than in offspring born to L. donovani-infected hamsters (8.5%, 3 of 35). After weaning, only offspring born to L. donovani-infected hamsters had lower weight gain (P < 0.001) and hematocrit levels (P = 0.0045) than controls. Challenge of offspring born to L. panamensis-infected hamsters with L. panamensis showed no differences in lesion evolution, and offspring born to L. donovani-infected hamsters were more susceptible to L. donovani challenge than controls. Consequently, prenatal exposure of hamsters to L. donovani significantly increased the mortality risk and susceptibility to secondary homologous infection.

Identification of small molecule lead compounds for visceral leishmaniasis using a novel ex vivo splenic explant model system.

BACKGROUND: New drugs are needed to treat visceral leishmaniasis (VL) because the current therapies are toxic, expensive, and parasite resistance may weaken drug efficacy. We established a novel ex vivo splenic explant culture system from hamsters infected with luciferase-transfected Leishmania donovani to screen chemical compounds for anti-leishmanial activity. METHODOLOGY/PRINCIPAL FINDINGS: THIS MODEL HAS ADVANTAGES OVER IN VITRO SYSTEMS IN THAT IT: 1) includes the whole cellular population involved in the host-parasite interaction; 2) is initiated at a stage of infection when the immunosuppressive mechanisms that lead to progressive VL are evident; 3) involves the intracellular form of Leishmania; 4) supports parasite replication that can be easily quantified by detection of parasite-expressed luciferase; 5) is adaptable to a high-throughput screening format; and 6) can be used to identify compounds that have both direct and indirect anti-parasitic activity. The assay showed excellent discrimination between positive (amphotericin B) and negative (vehicle) controls with a Z' Factor >0.8. A duplicate screen of 4 chemical libraries containing 4,035 compounds identified 202 hits (5.0%) with a Z score of <-1.96 (p<0.05). Eighty-four (2.1%) of the hits were classified as lead compounds based on the in vitro therapeutic index (ratio of the compound concentration causing 50% cytotoxicity in the HepG(2) cell line to the concentration that caused 50% reduction in the parasite load). Sixty-nine (82%) of the lead compounds were previously unknown to have anti-leishmanial activity. The most frequently identified lead compounds were classified as quinoline-containing compounds (14%), alkaloids (10%), aromatics (11%), terpenes (8%), phenothiazines (7%) and furans (5%). CONCLUSIONS/SIGNIFICANCE: The ex vivo splenic explant model provides a powerful approach to identify new compounds active against L. donovani within the pathophysiologic environment of the infected spleen. Further in vivo evaluation and chemical optimization of these lead compounds may generate new candidates for preclinical studies of treatment for VL.

Duplex real-time reverse transcriptase PCR to determine cytokine mRNA expression in a hamster model of New World cutaneous leishmaniasis.

BACKGROUND: The Syrian hamster, Mesocricetus auratus, has distinct immunological features and is uniquely susceptible to intracellular pathogens. Studies in hamsters are limited by the relative unavailability of tools to conduct immunological studies. To address this limitation we developed duplex real-time reverse transcriptase (RT) PCR assays for the relative quantification of the mRNAs of hamster cytokines, chemokines, and related immune response molecules. RESULTS: Real-time RT-PCR primers and probes were synthesized for analysis of interleukin (IL)-4, IFN-gamma, TNF-alpha, IL-10, IL-12p40, TGF-beta, IL-13, IL-21, chemokine ligand (CCL) 22, CCL17, Chemokine (C-C motif) receptor 4 and FoxP3 expression. Standard curves and validation experiments were performed for each real-time RT-PCR assay, allowing us to use the comparative Ct (2-DeltaDeltaCt) method to calculate changes in gene expression. Application of the real-time RT PCR assays to a biological model was demonstrated by comparing mRNA expression in skin and lymph node tissues between uninfected and Leishmania panamensis infected hamsters. CONCLUSIONS: The duplex real-time RT PCR assays provide a powerful approach for the quantification of cytokine transcription in hamsters, and their application to a model of cutaneous leishmaniasis suggests that a balanced type 1 and type 2 cytokine response contributes to the chronic, nonprogressive course of disease. These new molecular tools will further facilitate investigation into the mechanisms of disease in the hamster, not only for models of leishmaniasis, but also for other viral, bacterial, fungal, and parasitic infections.

Pregnancy enhances the innate immune response in experimental cutaneous leishmaniasis through hormone-modulated nitric oxide production.

The maintenance of host defense during pregnancy may depend on heightened innate immunity. We evaluated the immune response of pregnant hamsters during early infection with Leishmania (Viannia) panamensis, a cause of American cutaneous leishmaniasis. At 7 days post-infection, pregnant animals showed a lower parasite burden compared with nonpregnant controls at the cutaneous infection site (P=0.0098) and draining lymph node (P=0.02). Resident peritoneal macrophages and neutrophils from pregnant animals had enhanced Leishmania killing capacity compared with nonpregnant controls (P=0.018 each). This enhanced resistance during pregnancy was associated with increased expression of inducible NO synthase (iNOS) mRNA in lymph node cells (P=0.02) and higher NO production by neutrophils (P=0.0001). Macrophages from nonpregnant hamsters infected with L. panamensis released high amounts of NO upon estrogen exposure (P=0.05), and addition of the iNOS inhibitor L-N6-(1-iminoethyl) lysine blocked the induction of NO production (P=0.02). Infected, nonpregnant females treated with estrogen showed a higher percentage of cells producing NO at the infection site than controls (P=0.001), which correlated with lower parasite burdens (P=0.036). Cultured macrophages or neutrophils from estrogen-treated hamsters showed significantly increased NO production and Leishmania killing compared with untreated controls. iNOS was identified as the likely source of estrogen-induced NO in primed and naïve macrophages, as increased transcription was evident by real-time PCR. Thus, the innate defense against Leishmania infection is heightened during pregnancy, at least in part as a result of estrogen-mediated up-regulation of iNOS expression and NO production.

Pathogenicity and protective immunogenicity of cysteine proteinase-deficient mutants of Leishmania mexicana in non-murine models.

This study demonstrates that deletion of cysteine proteinase (CP) genes diminishes pathogenicity of Leishmania mexicana in non-murine experimental host models while preserving immunogenicity. Both cpb and cpa/cpb-deficient lines induced delayed disease onset, smaller lesions and lower parasite burden in hamsters. cpa/cpb-deficient L. mexicana grew more slowly as promastigotes and presented lower infectivity and growth in human mononuclear phagocytic host cells. Protection against homologous challenge comparable to that induced by infection with the virulent wild-type (WT) L. mexicana strain was achieved in the highly susceptible hamster model by immunization with 1000 cpb-deficient promastigotes. CP-deficient L. mexicana elicited significantly lower levels of Th2-associated cytokines IL-10 and TGF-beta than the WT in the primary lesion of hamsters. These findings support the feasibility of using genetically attenuated live Leishmania to achieve protective immunity.

Eficacia y toxicidad de los antimoniales pentavalentes (Glucantime® y Pentostam®) en un modelo animal de leishmaniasis cutánea americana: aplicación de la luminometría / Efficacy and toxicity of pentavalent antimonials (Glucantime® and Pentostam®) in an American cutaneous leishmaniasis animal model: luminometry application

Los antimoniales pentavalentes Glucantime® y Pentostam® son los medicamentos de primera línea usados en el tratamiento anti- Leishmania; sin embargo, no hay estudios in vivo que comparen su eficacia y toxicidad controlando variables del hospedero. Los estudios bioquímicos en Leishmania detectaron diferencias entre los dos medicamentos en la inhibición de la topoisomerasa I, que podrían reflejarse en diferencias en su efectividad. Para evaluar la eficacia clínica se infectaron hámsteres en la pata trasera derecha con 106 promastigotes de Leishmania panamensis silvestre y transfectada con el gen de la luciferasa. Para evaluar la eficacia parasitológica se estandarizó la cuantificación de parásitos en los tejidos por luminometría. Tres semanas después de la inoculación, los animales se trataron intramuscularmente con Glucantime® o Pentostam® (30, 60 o 120 mg de SbV/kg por día por 20 días). La toxicidad se evaluó en hámsteres tratados intramuscularmente con 120, 160 o 240 mg de SbV/kg por día por 20 días de Glucantime® o Pentostam®. La resolución de las lesiones en los animales inoculados con ambas cepas fue similar con ambos medicamentos. La carga parasitaria disminuyó de forma equivalente con ambos medicamentos en todas las dosis, y resultó en diferencias significativas con respecto a los controles ( p<0,01). Los niveles séricos de creatinina, aspartato aminotransferasa, alanino aminotransferasa y amilasa no evidenciaron alteraciones hepáticas o renales. Los animales tratados con dosis iguales o mayores de 120 mg de SbV/kg por día por 20 días de Pentostam® presentaron alteraciones inflamatorias micro y macroscópicas en el sitio de la inyección que no se observaron en los tratados con Glucantime®. Estos resultados confirman observaciones clínicas sobre la eficacia similar de ambos productos e indican una toxicidad local mayor del Pentostam®

[Efficacy and toxicity of pentavalent antimonials (Glucantime and Pentostam) in an American cutaneous leishmaniasis animal model: luminometry application]. / Eficacia y toxicidad de los antimoniales pentavalentes (Glucantime y Pentostam) en un modelo animal de leishmaniasis cutánea americana: aplicación de la luminometría.

The pentavalent antimonial compounds Glucantime and Pentostam are the first line drugs used in anti-Leishmania treatment. However, no in vivo studies have compared the efficacy and toxicity of these drugs where host variability has been controlled. Biochemical studies of Leishmania have detected differences between the two drugs with regard to DNA topoisomerase I inhibition, a phenomenon that possibly impacts treatment efficacy. To evaluate the clinical efficacy, hamsters were infected intradermally in the right hind foot with 10(6) promastigotes of a wild type or luciferase-transfected Leishmania panamensis. At three weeks post-inoculation, the animals were treated intramuscularly with either Glucantime or Pentostam (30, 60 or 120 mg SbV/kg per day for 20 days). To evaluate parasitological efficacy a luminometry assay was standardized for quantitation of amastigotes in hamster tissues. To evaluate toxicity, hamsters were treated intramuscularly with Glucantime or Pentostam (120, 160 or 240 mg SbV/kg per day for 20 days). Animals inoculated with either of the parasite strains and treated with either drug, showed a similar rate of lesion reduction, as compared to untreated controls (p<0.01). Parasite burden was also comparable, and no significant differences were found in the cure rate. No renal or hepatic alterations occurred as evidenced by normal serum levels of creatinine, aspartate aminotransferase, alanine aminotransferase and amylase. Hamsters treated with 120 mg SbV/kg per day for 20 days or higher doses of Pentostam showed macro- and microscopic signs of inflammation at the site of injection. These effects were absent in the animals treated with Glucantime. The results confirmed clinical observations regarding the similar efficacy of the two drugs, as well as the higher local toxicity of Pentostam.

The site of cutaneous infection influences the immunological response and clinical outcome of hamsters infected with Leishmania panamensis.

We determined that the site of inoculation (foot or snout) influences the clinical evolution and immune responses of hamsters infected with Leishmania (Viannia) panamensis. Hamsters infected in the snout showed (i) a more rapid and severe lesion evolution at multiple time points (P < 0.05), (ii) a more extensive inflammatory infiltrate and tissue necrosis, (iii) a higher tissue parasite burden, (iv) a higher antibody titre (P < 0.01), but lower antigen-specific spleen cell proliferative response (P = 0.02), and (v) a slower response to anti-leishmanial drug treatment (P < 0.002). In both inoculation groups there was co-expression of type 1 (IFN-gamma and IL-12) and some type 2 (IL-10 and TGF-beta, but not IL-4) cytokines in the cutaneous lesions and spleen. Early in the course of infection, hamsters infected in the snout showed higher expression of splenic IL-10 (P = 0.04) and intra-lesional IFN-gamma (P = 0.02) than foot infections. No expression of IL-12p40 or IL-4 was detected. During the chronic phase, snout lesions expressed more IFN-gamma (P = 0.001), IL-12p40 (P = 0.01), IL-10 (P = 0.009) and TGF-beta (P = 0.001), and the level of expression of each of these cytokines correlated with lesion size (P < or = 0.01). These results suggest that the site of infection influences the clinical outcome in experimental cutaneous leishmaniasis, and that the expression of macrophage-deactivating type 2 cytokines and/or an exaggerated type 1 proinflammatory cytokine response may contribute to lesion severity.

Gender is a major determinant of the clinical evolution and immune response in hamsters infected with Leishmania spp.

In regions where leishmaniasis is endemic, clinical disease is usually reported more frequently among males than females. This difference could be due to disparate risks of exposure of males and females, but gender-related differences in the host response to infection may also play a role. Experimental studies of the influence of gender on Leishmania infection have not included parasites of the subgenus Viannia, which is the most common cause of cutaneous leishmaniasis in the Americas. Mice are not readily susceptible to infection by Leishmania (Viannia) spp., but cutaneous infection of hamsters with L. (V.) panamensis or L. (V.) guyanensis resulted in chronic lesions typical of the human disease caused by these parasites. Strikingly, infection of male hamsters resulted in significantly greater lesion size and severity, an increased rate of dissemination to distant cutaneous sites, and a greater parasite burden in the draining lymph node than infection in female animals. Two lines of evidence indicated this gender-related difference in disease evolution was determined at least in part by the sex hormone status of the animal. First, prepubertal male animals had smaller and/or less severe cutaneous lesions than adult male animals. Second, infection of testosterone-treated female animals resulted in significantly larger lesions than in untreated female animals. The increased severity of disease in male compared to female animals was associated with significantly greater intralesional expression of interleukin-4 (IL-4) (P = 0.04), IL-10 (P = 0.04), and transforming growth factor beta (TGF-beta) (P < 0.001), cytokines known to promote disease in experimental leishmaniasis. There was a direct correlation between the expression of TGF-beta mRNA and lesion size (Spearman's correlation coefficient = 0.873; P < 0.001). These findings demonstrate an inherent risk of increased disease severity in male animals, which is associated with a more permissive immune response.

Resumen del taller sobre el uso de la reacción en cadena de la polimerasa (PCR) para distinguir entre Trypanosoma cruzi y Trypanosoma rangeli / Summary of the workshop: use of the polymerase chain reaction (PCR) to differentiate Trypanosoma cruzi from Trypanosoma rangeli

Este taller se realizó con el propósito de transferir la tecnología de la reacción en cadena de la polimerasa (PCR) para la detección de T. cruzi y T. rangeli a laboratorios en Colombia involucrados en el diagnóstico y estudios epidemiológicos de la enfermedad de Chagas. Para demostración de la técnica se utilizaron muestras clínicas y epidemiológicas de áreas endémicas colombianas. En los ensayos se emplearon muestras de tripanosomas provenientes de diferentes medios de cultivo para evaluar el posible efecto de los componentes de estos medios sobre la sensibilidad de PCR. Se hizo extracción de ADN utilizando los métodos de ebullición, lisis hipotónica y geneclean. El ADN se amplificó utilizando oligonucleótidos sintéticos, correspondientes a una secuencia conservada de 22 nucleótidos dentro de un gen mini-exón. Los dos organismos fueron distinguidos por las movilidades electroforéticas de sus respectivos productos de amplificación, confirmando su identidad con sondas intergénicas específicas de especie, marcadas con digoxigenina dUTP. La hibridación se visualizó con la reacción de color del NBT. De un total de 28 muestras analizadas, se lograron 17 identificaciones que coincidieron con la clasificación original. De cinco muestras desconocidas, tres fueron identificadas como T. rangeli y dos como infecciones mixtas. Se presentaron resultados ambiguos en dos muestras, ocasionados por contaminación en PCR. Solamente dos muestras no se pudieron identificar mediante PCR por problemas en la extracción del ADN de la muestra. Teniendo en cuenta estos resultados preliminares se abre la posibilidad de utilizar esta técnica como una herramienta útil o método adicional a las técnicas de rutina para detectar y diagnósticar la enfermedad de Chagas en Colombia