Dilema ético y epidemiológico sobre el tratamiento de perros para la leishmaniasis visceral en América Latina

In the Americas there are between 4,500 and 6,800 annual cases of severe visceral leishmaniasis, and mortality is estimated to range between 7 and 10%. However, underreporting and subclinical infections mask the real epidemiological importance of visceral leishmaniasis. Control efforts, which have typically focused on insecticide spraying of sand fly vectors and dog culling, have yielded disparate results. Nevertheless, thousands of dogs are sacrificed each year in countries endemic for visceral leishmaniasis. Additionally, current guidelines of leishmaniasis control programs have banned dog treatment with drugs of human use while therapy with other drugs resulted in high rates of relapses. Society requires that control programs take a more humanitarian approach aimed at limiting dog culling. There is an urgent need to promote responsible dog-ownership and support research on: a) novel veterinary therapies, b) low-cost molecular diagnosis of canine visceral leishmaniasis, and c) determination of dog infectivity threshold for proper reservoir management.En las Américas hay entre 4.500 y 6.800 casos anuales de leishmaniasis visceral grave y se estima que la mortalidad varía entre 7 y 10 %. Sin embargo, el subregistro y las infecciones subclínicas enmascaran la importancia epidemiológica real de la leishmaniasis visceral. Los esfuerzos de control que típicamente se han enfocado en la aspersión de insecticidas contra los flebotomíneos vectores y el sacrificio de perros, han arrojado resultados dispares. No obstante, miles de perros se sacrifican cada año en países endémicos para leishmaniasis visceral. Además, los lineamientos actuales de los programas de control de la leishmaniasis han prohibido el tratamiento de perros con medicamentos de uso humano, mientras que otras drogas resultan en altas tasas de recaída. La sociedad requiere que los programas de control tengan un manejo más humanitario enfocado a limitar el sacrificio canino. Hay una necesidad urgente de promover la tenencia responsable de los perros y apoyar la investigación en: a) terapias veterinarias novedosas, b) diagnósticos moleculares de bajo costo y c) determinación de los umbrales de capacidad infecciosa canina para el manejo adecuado del reservorio.

Cuantificación de citocinas caninas mediante reacción en cadena de la polimerasa de transcriptasa reversa en tiempo real

Introduction. Canines are the principal domestic reservoirs of visceral leishmaniasis in both the Old and New World. The development of highly sensitive and quantitative methods, such as real time reverse transcriptase polymerase chain reaction for measurement of canine cytokines has not been exploited in studies of visceral leishmaniasis.Objective. To standardize the relative quantification of canine IFN-g, IL-4, IL-10, IL-12p40 and IL-12p35 using real time reverse transcriptase polymerase chain reaction.Materials and methods. RNA was isolated from PBMCs from 1 year–old foxhounds and cultured with or without Con A, LPS orStaphylococcus aureus extract. This RNA was used in one-step real time reverse transcriptase polymerase chain reaction to optimize the concentrations of the cytokine primers and probes, generate standard curves for each cytokine, confirm equivalent amplification efficiency of cytokine and normalizer (18S rRNA) RNA, and to quantify the expression of the cytokine RNA. The comparative Ct method was used to determine the relative levels of gene expression in the samples, expressed as the fold-increase relative to the control samples.Results. The regression coefficient for the standard curves and the amplification efficiencies of the cytokine and normalizer RNA indicated that the quantification was reliable over a broad concentration range of input RNA. Relative to control cells, activation of PBMCs led to increased expression of IFN-g (132-fold), IL-4 (8.8-fold), IL-10 (7.2-fold), and IL-12p40 (275-fold). Basal expression of IL-12p35 was also detected.Conclusion. This approach provides several advantages over conventional assays for cytokine measurement and can be exploited in the study of the immunopathogenesis and immunity in canine leishmaniasis.Introducción. Los caninos son el principal reservorio domestico de la leishmaniasis visceral en el Nuevo y Viejo mundo. La reacción en cadena de la polimerasa de transcriptasa reversa en tiempo real para la medición de citocinas caninas no ha sido implementada para el estudio de la leishmaniasis visceral.Objetivo. Estandarizar la cuantificación relativa de IFN-g, IL-4, IL-10, IL-12p40 y IL-12p35 caninas utilizando reacción en cadena de la polimerasa de transcriptasa reversa en tiempo real.Materiales y métodos. Células mononucleares de sangre periférica de perros Fox-Hound fueron estimuladas con ConA, LPS y extracto de Staphylococcus aureus. El ARN fue utilizado en la reacción en cadena de la polimerasa de transcriptasa reversa en tiempo real de un solo paso para optimizar las concentraciones de iniciadores y sondas especificas de cada citocina, generar curvas estándar, confirmar la eficiencia de amplificación de las citocinas y del normalizador (18S ARNr) y cuantificar la expresión de ARN. El método comparativo Ct fue utilizado para determinar los niveles relativos de expresión de ARN en las muestras, expresado como el incremento en el número de veces comparado con los controles.Resultados. El coeficiente de regresión para las curvas estándar y las eficiencias de amplificación de las citocinas y el normalizador, indicaron que la cuantificación fue confiable en un amplio rango de concentraciones de ARN. La activación de células mononucleares de sangre periférica resultó en un incremento en la expresión de IFN-g (132), IL-4 (8.8), IL-10 (7,2), y IL-12p40 (275), relativo a células control. La expresión basal de IL-12p35 fue también detectada.Conclusión. Esta metodología, comparada con los métodos convencionales disponibles para la medición de citocinas, ofrece varias ventajas y podría ser utilizada en estudios sobre inmunopatogenia e inmunidad en leishmaniasis visceral canina

Éxito de la alimentación de Lutzomyia evansi (Diptera: Psychodidae) expuestos experimentalmente a reservorios mamíferos pequeños en un foco endémico de Leishmania chagasi en el norte de Colombia

Lutzomyia evansi is the vector of Leishmania chagasi in northern Colombia. Differences in feeding success were revealed, when this phlebotomine sand fly was fed on five species of small mammal hosts from an endemic focus of visceral leishmaniasis. In each trial, 50 female sand flies were provided access to similar-sized depilated areas of the hind foot of each of 44 individual mammals and allowed to feed for 30 minutes. The number of engorged sand flies was counted at the end of each trial and compared among host species by analysis of variance and Tukey's multiple comparisons test. Sand flies fed least successfully on Sciurus granatensis, a common squirrel in the endemic area. It has not been found infected with L. chagasi. Intermediate numbers of sand flies engorged on Heteromys anomalus and Zygodontomys brevicauda, but these two mammals have not been found infected with L. chagasi and are not expected to be important in transmission. Sand flies fed most successfully on Didelphis marsupialis and Proechimys canicollis. These are the two most abundant mammals in the endemic area and frequently are infected. Results provided further evidence that these two species are the wild mammals with the greatest impact on transmission of L. chagasi in northern Colombia.Un método sencillo de laboratorio reveló diferencias en el éxito de alimentación de Lutzomyia evansi, el vector de Leishmania chagasi en el norte de Colombia, cuando se alimentó sobre cinco especies de pequeños mamíferos de un foco endémico de leishmaniasis visceral, en los que éstos podrían actuar como reservorios. En cada ensayo, a 50 flebótomos hembra se les permitió alimentarse durante 30 minutos sobre un área similar de piel depilada de la pata posterior en 44 mamíferos. El número de flebótomos alimentados se comparó entre especies a través de un análisis de varianza y de la prueba de Tukey de comparaciones múltiples. Los flebótomos escasamente se alimentaron sobre Sciurus granatensis, una ardilla común en el área endémica que no se ha encontrado infectada con Leishmania chagasi. En otros dos mamíferos que hasta el presente han sido negativos para L. chagasi (Heteromys anomalus y Zygodontomys brevicauda), los flebótomos se alimentaron en bajos números. En cambio, los flebótomos se alimentaron en altas proporciones sobre Didelphis marsupialis y Proechimys canicollis, los dos mamíferos más abundantes en el área endémica y que se hallan infectados con L. chagasi. Los resultados aportaron evidencia adicional que estas dos especies de mamíferos silvestres serían una fuente de sangre común para los flebótomos y que, por lo tanto, pueden tener gran impacto sobre la transmisión de L. chagasi en el norte de Colombia

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

<p>Abstract</p> <p>Background</p> <p>The Syrian hamster, <it>Mesocricetus auratus</it>, 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.</p> <p>Results</p> <p>Real-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 <it>Leishmania panamensis </it>infected hamsters.</p> <p>Conclusions</p> <p>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.</p

¿Oncocercosis en Colombia? Una revaluación del foco de López de Micay

Human onchocerciasis was first described as existing in Colombia in 1965 in a small focus in Lopez de Micay on the Pacific Coast. Subsequent follow-up of the focus 12 years later showed that infection-prevalence had fallen from 15 to 7.5%. Since no patients were reported thereafter, the focus was considered to have been extinguished until 1989, when a child with occular keratitis was referred to the University Hospital in Cali. Onchocerciasis was confirmed by skin,snip examination. In July 1989 a multidisciplinaty team conducted a new survey in the area. Skin biopsies were obtained from 170 individuals. Infection-prevalence detected by skin-snip examination was 4.1% (71170). Ten percent of the surveyed males and 0.9% of the females had detectable microfilariae (mf) in skin. Microfilariae density in skin varied between 0.5 mf per milligram of skin to 47 mflmg and was directly related to the patients' age. Neither palpable subcutaneous nodules nor dermal alterations attributable to the parasite were detected in any of the patients. Occular pathology was found in two patients, consisting of bilateral keratitis and retinal degeneration, respectively. Simuliid activity at the time of the survey was very low and collection was not attempted. We hypothesise that active transmission may be taking place further upstream, where vector activity is greater. An increase in non-immune human settlement in the area is expected, due to the future construction of hydroelectrical plants and related connecting highways. Questions are therefore raised concerning the impact of immigration of naive population to this hypoendemic focus.En 1965 se describió el primer foco de oncocercosis humana en Colombia en López de Micay, en la costa pacífica. Doce años después, una visita de seguimiento mostró que la prevalencia de infección había disminuido del 15 al 7,5%. Ya que no se volvieron a reportar pacientes, se consideró extinguido el foco hasta 1989, cuando un niño con queratitis ocular fue remitido al Hospital Universitario de Cali. Se confirmó el diagnóstico de oncocercosis con una biopsia de piel. En julio de 1989 se hizo una nueva visita al área y se tomaron muestras a 170 personas. La prevalencia de infección detectada por biopsia de piel fue de 4,1% (71170). Se detectaron microfilarias (mf) en piel en 10% de los hombres muestreados y 0,9% de las mujeres. La microfilarodermia varió entre 0,5 mf por miligramo de piel y 47mflmg y estaba directamente relacionada con la edad. No se encontraron nódulos subcutáneos palpables ni alteraciones dérmicas atribuibles al parásito en ningún paciente. Dos pacientes presentaron cambios oculares: queratitis bilateral y degeneración de la retina, respectivamente. Durante la visita, la actividad de los simúlidos fue muy baja y no se intentaron capturas. Es probable que la transmisión ocurra en sitios río arriba, donde la actividad de los vectores es mayor. Con la construcción de vías de acceso y una planta hidroeléctrica se espera un aumento de la población no inmune en el área. Cabe preguntar, cúal será el impacto de esta población susceptible sobre este foco hipoendémico

Inflammatory stimuli alter bone marrow composition and compromise bone health in the malnourished host

Inflammation has a role in the pathogenesis of childhood malnutrition. We investigated the effect of malnutrition and inflammatory challenge on bone marrow composition and bone health. We studied an established murine model of moderate acute malnutrition at baseline and after acute inflammatory challenge with bacterial lipopolysaccharide (LPS), a surrogate of Gram-negative bacterial sepsis, or Leishmania donovani, the cause of visceral leishmaniasis. Both of these infections cause significant morbidity and mortality in malnourished children. Of the 2 stimuli, LPS caused more pronounced bone marrow changes that were amplified in malnourished mice. LPS challenge led to increased inflammatory cytokine expression (Il1b, Il6, and Tnf), inflammasome activation, and inflammatory monocyte accumulation in the bone marrow of malnourished mice. Depletion of inflammatory monocytes in Csfr1-LysMcre-DT malnourished mice significantly reduced the inflammasome activation and IL1-ß production after LPS challenge. The inflammatory challenge also led to increased expansion of mesenchymal stem cells (MSCs), bone marrow adiposity, and expression of genes (Pparg, Adipoq, and Srbp1) associated with adipogenesis in malnourished mice. This suggests that inflammatory challenge promotes differentiation of BM MSCs toward the adipocyte lineage rather than toward bone-forming osteoblasts in the malnourished host. Concurrent with this reduced osteoblastic potential there was an increase in bone-resorbing osteoclasts, enhanced osteoclast activity, upregulation of inflammatory genes, and IL-1B involved in osteoclast differentiation and activation. The resulting weakened bone formation and increased bone resorption would contribute to the bone fragility associated with malnutrition. Lastly, we evaluated the effect of replacing lipid rich in omega-6 fatty acids (corn oil) with lipid-rich in omega-3 fatty acids (fish oil) in the nutrient-deficient diet. LPS-challenged malnourished mice that received dietary fish oil showed decreased expression of inflammatory cytokines and Rankl and reduced osteoclast differentiation and activation in the bone marrow. This work demonstrates that the negative effect of inflammatory challenge on bone marrow is amplified in the malnourished host. Increasing dietary intake of omega-3 fatty acids may be a means to reduce inflammation and improve bone health in malnourished children

Pathologic Inflammation in Malnutrition Is Driven by Proinflammatory Intestinal Microbiota, Large Intestine Barrier Dysfunction, and Translocation of Bacterial Lipopolysaccharide

Acute malnutrition, or wasting, is implicated in over half of all deaths in children under five and increases risk of infectious disease. Studies in humans and preclinical models have demonstrated that malnutrition is linked to an immature intestinal microbiota characterized by increased prevalence of Enterobacteriaceae. Observational studies in children with moderate acute malnutrition (MAM) have also observed heightened systemic inflammation and increased circulating bacterial lipopolysaccharides (LPS; endotoxin). However, the mechanisms that underpin the systemic inflammatory state and endotoxemia, and their pathophysiological consequences, remain uncertain. Understanding these pathophysiological mechanisms is necessary to design targeted treatments that will improve the unacceptable rate of failure or relapse that plague current approaches. Here we use a mouse model of MAM to investigate the mechanisms that promote inflammation in the malnourished host. We found that mice with MAM exhibited increased systemic inflammation at baseline, increased translocation of bacteria and bacterial LPS, and an exaggerated response to inflammatory stimuli. An exaggerated response to bacterial LPS was associated with increased acute weight loss. Remarkably, intestinal inflammation and barrier dysfunction was found in the cecum and colon. The cecum showed a dysbiotic microbiota with expansion of Gammaproteobacteria and some Firmicutes, and contraction of Bacteroidetes. These changes were paralleled by an increase in fecal LPS bioactivity. The inflammatory phenotype and weight loss was modulated by oral administration of non-absorbable antibiotics that altered the proportion of cecal Gammaproteobacteria. We propose that the heightened inflammation of acute malnutrition is the result of changes in the intestinal microbiota, intestinal barrier dysfunction in the cecum and colon, and increased systemic exposure to LPS

Identification of Small Molecule Lead Compounds for Visceral Leishmaniasis Using a Novel Ex Vivo Splenic Explant Model System

Visceral leishmaniasis is a life threatening parasitic disease present in several countries of the world. New drugs are needed to treat this disease because treatments are becoming increasingly ineffective. We established a novel system to screen for new anti-leishmanial compounds that utilizes spleen cells from hamsters infected with the parasite Leishmania donovani. The parasite strain we used was genetically engineered to emit light by the incorporation of the firefly luciferase gen. This laboratory test system has the advantage of reproducing the cellular environment where the drug has to combat the infection. The efficacy of the compounds is easily determined by measuring the light emitted by the surviving parasites in a luminometer after exposing the infected cells to the test compounds. The screening of more than 4,000 molecules showed that 84 (2.1%) of them showed anti-leishmanial activity and had an acceptable toxicity evaluation. Eighty two percent of these molecules, which had varied chemical structures, were previously unknown to have anti-leishmanial activity. Further studies in animals of these new chemical entities may identify drug candidates for the treatment of visceral leishmaniasis

Progressive Visceral Leishmaniasis Is Driven by Dominant Parasite-induced STAT6 Activation and STAT6-dependent Host Arginase 1 Expression

The clinicopathological features of the hamster model of visceral leishmaniasis (VL) closely mimic active human disease. Studies in humans and hamsters indicate that the inability to control parasite replication in VL could be related to ineffective classical macrophage activation. Therefore, we hypothesized that the pathogenesis of VL might be driven by a program of alternative macrophage activation. Indeed, the infected hamster spleen showed low NOS2 but high arg1 enzyme activity and protein and mRNA expression (p<0.001) and increased polyamine synthesis (p<0.05). Increased arginase activity was also evident in macrophages isolated from the spleens of infected hamsters (p<0.05), and arg1 expression was induced by L. donovani in primary hamster peritoneal macrophages (p<0.001) and fibroblasts (p<0.01), and in a hamster fibroblast cell line (p<0.05), without synthesis of endogenous IL-4 or IL-13 or exposure to exogenous cytokines. miRNAi-mediated selective knockdown of hamster arginase 1 (arg1) in BHK cells led to increased generation of nitric oxide and reduced parasite burden (p<0.005). Since many of the genes involved in alternative macrophage activation are regulated by Signal Transducer and Activator of Transcription-6 (STAT6), and because the parasite-induced expression of arg1 occurred in the absence of exogenous IL-4, we considered the possibility that L. donovani was directly activating STAT6. Indeed, exposure of hamster fibroblasts or macrophages to L. donovani resulted in dose-dependent STAT6 activation, even without the addition of exogenous cytokines. Knockdown of hamster STAT6 in BHK cells with miRNAi resulted in reduced arg1 mRNA expression and enhanced control of parasite replication (p<0.0001). Collectively these data indicate that L. donovani infection induces macrophage STAT6 activation and STAT6-dependent arg1 expression, which do not require but are amplified by type 2 cytokines, and which contribute to impaired control of infection