Yaneth Osorio

Gender Is a Major Determinant of the Clinical Evolution and Immune Response in Hamsters Infected with Leishmania spp.

ABSTRACT 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 β (TGF-β) (P < 0.001), cytokines known to promote disease in experimental leishmaniasis. There was a direct correlation between the expression of TGF-β mRNA and lesion size (Spearmans 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.

Dynamics of Leishmania chagasi infection in small mammals of the undisturbed and degraded tropical dry forests of northern Colombia

The infection rate with Leishmania chagasi and the population dynamics of small mammals were studied in an undisturbed forest reserve (Colosó) and an area of highly degraded forest (San Andrés de Sotavento [SAS]) in northern Colombia, both endemic for visceral leishmaniasis. Live trapping of mammals was done every month, and species, age, sex and reproductive status determined. L. chagasi was detected in samples of skin or spleen by the polymerase chain reaction, after extraction of deoxyribonucleic acid using specific primers (DB8/AJS3), and dot blood hybridization. Didelphis marsupialis was found to be infected in Colosó (3/21, 14.3%) and SAS (13/137, 9.5%); its relative abundance was higher in SAS (93/113, 82% of the captures). Although Proechimys canicollis was also found to be infected in Colosó (3/34, 8.8%) and SAS (2/4), its relative abundance was much lower (4%) in SAS than in Colosó (56% of 77 animals captured). Sciurus granatensis, Marmosa robinsoni, Heteromys anomalus, Zygodontomys brevicauda and Metachirus nudicaudatus were less common, and no L. chagasi infection was detected in them.

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.

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‐γ and IL‐12) and some type 2 (IL‐10 and TGF‐β, 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‐γ (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‐γ (P = 0·001), IL‐12p40 (P = 0·01), IL‐10 (P = 0·009) and TGF‐β (P = 0·001), and the level of expression of each of these cytokines correlated with lesion size (P ≤ 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.

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

BackgroundThe 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.ResultsReal-time RT-PCR primers and probes were synthesized for analysis of interleukin (IL)-4, IFN-γ, TNF-α, IL-10, IL-12p40, TGF-β, 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-ΔΔCt) 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.ConclusionsThe 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.

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.

Development of an Ex Vivo Lymph Node Explant Model for Identification of Novel Molecules Active against Leishmania major

ABSTRACT 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.