Resistance to Oxidative Stress Is Associated with Metastasis in Mucocutaneous Leishmaniasis

Mucocutaneous leishmaniasis (MCL) in South and Central America is characterized by the dissemination (metastasis) of Leishmania Viannia subgenus parasites from a cutaneous lesion to nasopharyngeal tissues. Little is known about the pathogenesis of MCL, especially with regard to the virulence of the parasites and the process of metastatic dissemination. We previously examined the functional relationship between cytoplasmic peroxiredoxin and metastatic phenotype using highly, infrequently, and nonmetastatic clones isolated from an L. (V.) guyanensis strain previously shown to be highly metastatic in golden hamsters. Distinct forms of cytoplasmic peroxiredoxin were identified and found to be associated with the metastatic phenotype. We report here that peroxidase activity in the presence of hydrogen peroxide and infectivity differs between metastatic and nonmetastatic L. (V.) guyanensis clones. After hydrogen peroxide treatment or heat shock, peroxiredoxin was detected preferentially as dimers in metastatic L. (V.) guyanensis clones and in L. (V.) panamensis strains from patients with MCL, compared with nonmetastatic parasites. These data provide evidence that resistance to the first microbicidal response of the host cell by Leishmania promastigotes is linked to peroxiredoxin conformation and may be relevant to intracellular survival and persistence, which are prerequisites for the development of metastatic diseas

Mapa del proteoma de parásitos de Leishmania Viannia a partir de electroforesis de geles de poliacramida en dos dimensiones y otras técnicas asociadas

In this study we have demonstrated the potential of two-dimensional electrophoresis (2DE)-based technologies as tools for characterization of the Leishmania proteome (the expressed protein complement of the genome). Standardized neutral range (pH 5-7) proteome maps of Leishmania (Viannia) guyanensis and Leishmania (Viannia) panamensis promastigotes were reproducibly generated by 2DE of soluble parasite extracts, which were prepared using lysis buffer containing urea and nonidet P-40 detergent. The Coomassie blue and silver nitrate staining systems both yielded good resolution and representation of protein spots, enabling the detection of approximately 800 and 1,500 distinct proteins, respectively. Several reference protein spots common to the proteomes of all parasite species/strains studied were isolated and identified by peptide mass spectrometry (LC-ES-MS/MS), and bioinformatics approaches as members of the heat shock protein family, ribosomal protein S12, kinetoplast membrane protein 11 and a hypothetical Leishmania-specific 13 kDa protein of unknown function. Immunoblotting of Leishmania protein maps using a monoclonal antibody resulted in the specific detection of the 81.4 kDa and 77.5 kDa subunits of paraflagellar rod proteins 1 and 2, respectively. Moreover, differences in protein expression profiles between distinct parasite clones were reproducibly detected through comparative proteome analyses of paired maps using image analysis software. These data illustrate the resolving power of 2DE-based proteome analysis. The production and basic characterization of good quality Leishmania proteome maps provides an essential first step towards comparative protein expression studies aimed at identifying the molecular determinants of parasite drug resistance and virulence, as well as discovering new drug and vaccine targets.En este estudio demostramos el potencial de la electroforesis en dos dimensiones (2DE) como herramienta para la caracterización del proteoma de Leishmania (expresión proteica complementaria del genoma). Los proteomas por 2DE en el rango neutro (pH 5-7) de extractos solubles de promastigotes de Leishmania (Viannia) guyanensis y Leishmania (Viannia) panamensis fueron reproducibles usando tampón de lisis de urea y nonidet P-40. Con la tinción de azul de Coomassie y nitrato de plata se detectaron, con buena resolución, 800 y 1.500 puntos de proteínas, respectivamente. Entre las proteínas de referencia comunes, aisladas de los proteomas de las cepas estudiadas, se identificaron por medio de espectrometría de masa de péptidos (LC-ES-MS/MS) y métodos bioinformáticos, proteínas de choque térmico, proteína ribosomal S12, proteína de membrana del cinetoplasto 11 y una proteína hipotética específica de Leishmania de 13 kDa con función desconocida. Por inmunoblot y utilizando un anticuerpo monoclonal, se detectaron específicamente las proteínas paraflagelar 1 y 2 de 81,4 kDa y 77,5 kDa, respectivamente. La expresión proteica diferencial encontrada en los distintos clones de parásitos fueron reproducibles al ser comparados por medio de un programa analizador de imágenes. Estos datos demuestran el poder de resolución del análisis de proteomas por 2DE. La producción y caracterización de mapas proteicos básicos de Leishmania con buena calidad constituye un paso esencial en los estudios proteómicos comparativos orientados a la identificación de factores moleculares involucrados en la resistencia a drogas, virulencia de parásitos y el descubrimiento de blancos para nuevas drogas y vacunas

Viability and Burden of Leishmania in Extralesional Sites during Human Dermal Leishmaniasis

Understanding of the dynamics and distribution of Leishmania in the human host is fundamental to the targeting of control measures and their evaluation. Amplification of parasite gene sequences in clinical samples from cutaneous leishmaniasis patients has provided evidence of Leishmania in blood, other tissues and sites distinct from the lesion and of persistence of infection after clinical resolution of disease. However, there is uncertainty about the interpretation of the presence of Leishmania DNA as indicative of viable parasites. Because RNA is short-lived and labile, its presence provides an indicator of viability. We amplified Leishmania 7SLRNA, a molecule involved in intracellular protein translocation, to establish viability and estimate parasite load in blood monocytes, tonsil swab samples, and tissue fluid from healthy skin of patients with dermal leishmaniasis. Results showed that during active dermal leishmaniasis, viable Leishmania are present in blood monocytes, tonsils and normal skin in quantities similar to that in lesions, demonstrating widespread dissemination of infection and subclinical involvement of tissues beyond the lesion site. Leishmania 7SLRNA will be useful in deciphering the role of human infection in transmission

Advances in leishmaniasis.

Governed by parasite and host factors and immunoinflammatory responses, the clinical spectrum of leishmaniasis encompasses subclinical (inapparent), localised (skin lesions), and disseminated infection (cutaneous, mucosal, or visceral). Symptomatic disease is subacute or chronic and diverse in presentation and outcome. Clinical characteristics vary further by endemic region. Despite T-cell-dependent immune responses, which produce asymptomatic and self-healing infection, or appropriate treatment, intracellular infection is probably life-long since targeted cells (tissue macrophages) allow residual parasites to persist. There is an epidemic of cutaneous leishmaniasis in Afghanistan and Pakistan and of visceral infection in India and Sudan. Diagnosis relies on visualising parasites in tissue or serology; culture and detection of parasite DNA are useful in the laboratory. Pentavalent antimony is the conventional treatment; however, resistance of visceral infection in India has spawned new treatment approaches--amphotericin B and its lipid formulations, injectable paromomycin, and oral miltefosine. Despite tangible advances in diagnosis, treatment, and basic scientific research, leishmaniasis is embedded in poverty and neglected. Current obstacles to realistic prevention and proper management include inadequate vector (sandfly) control, no vaccine, and insufficient access to or impetus for developing affordable new drugs

Eficacia de amodiaquina y sulfadoxina/pirimetamina en el tratamiento de malaria no complicada por Plasmodium falciparum en Nariño, Colombia, 1999-2002.

La resistencia a los antimaláricos es una de las causas del aumento de casos de malaria en el mundo. Desde el año 2000, el tratamiento de malaria no complicada por Plasmodium falciparum en Colombia ha sido la combinación de amodiaquina (AQ) y sulfadoxina/pirimetamina (SP). La eficacia de estos dos medicamentos se evaluó después de la implementación del nuevo esquema. El estudio se realizó en los municipios de El Charco y Tumaco (Nariño) en la Costa Pacífica. Se utilizó el protocolo estándar de la OPS para la evaluación de la eficacia de antimaláricos en áreas de baja a moderada transmisión. Los pacientes incluidos fueron asignados al azar a los dos medicamentos de estudio y seguidos hasta el día 14 en El Charco y hasta el día 28 en Tumaco. Ninguno de los 48 pacientes en El Charco presentó falla terapéutica a los medicamentos en estudio. En Tumaco, por el contrario, 12 de 24 pacientes (IC95%: 30,6 a 69,4) presentaron falla a la AQ y 4 de 26 (IC95%:5,1-33,1) presentaron falla a SP. Los altos niveles de falla a AQ en Tumaco fueron inesperados por su reciente introducción oficial al tratamiento de malaria, mientras que los niveles de falla a SP aumentaron respecto a lo encontrado en estudios anteriores. Estos hallazgos sugieren que el uso de AQ a las dosis actuales en combinación con SP tendrá un tiempo de vida útil más corto que el esperado. El uso combinado de antimaláricos como estrategia para retardar la aparición de resistencia será efectivo en la medida en que las monoterapias sean eficaces