Leishmania (Viannia) lainsoni: occurrence of intracellular promastigote forms in vivo and in vitro

Experimental chronic (45-day-old) skin lesion in hamster hind foot induced by Leishmania (Viannia) lainsoni infection showed the presence of promastigote forms in the tissue, inside parasitophorous vacuoles, as assessed by transmission electron microscopy. Experimental in vitro interaction (24 and 48 h) between Leishmania (V.)lainsoni and J774-G8 macrophage cells also demonstrated the same profile. This morphological aspect is unusual, since in this parasite genus only amastigote forms have been described as the resistant and obligate intracellular forms.

Avaliação morfológica de lesões ativas cutâneas humanas de leishmaniose tegumentar americana / Morphologic evaluation of active injuries cutaneous human beings of leishmaniasis diffuse cutaneous

O perfil celular no microambiente do granuloma tecidual de lesões cutâneas ativas de pacientes com LTA de áreas endêmicas do Estado do Rio de Janeiro, foi analisado por microscopia de luz convencional, aliada ao uso de técnicas de fluorescência (TUNEL), imunohistoquímica e microscopia eletrônica de transmissão convencional. A análise permitiu identificar alterações no perfil celular que variava de acordo com o tempo de infecção. Em lesões recentes (15 dias a 2 meses) de evolução, foi possível observar um número expressivo de neutrófilos, eosinófilos, linfócitos e alguns monócitos e macrófagos. Em lesões mais tardias (4-6 meses) de evolução, houve um aumento expressivo de células, particularmente e plasmócitos apresentando RER bem desenvolvido e algumas vesículas secretórias sugerindo intensa produção de imunoglobulinas e macrófagos. As lesões tardias com 6 meses de evolução apresentaram uma característica de destaque: grande quantidade de células em processo de morte celular como autofagia, necrose e apoptose. Por fluorescência (TUNEL), foi verificado um aumento crescente do número de células apoptóticas que variava de acordo com o tempo de infecção das lesões. A caracterização ultra-estrutural das lesões mostrou células como plasmócitos, em estágios progressivos de indicação de morte celular, sugerindo etapas iniciais de autofagia: (i) perda da arquitetura normal do RER, com arredondamento de suas cisternas, algumas vezes mostrando dilatação excessiva e ainda, aumento do espaço intermembranar do envoltório nuclear (ii) ausência de Golgi e grande quantidade de vesículas secretórias com conteúdo rarefeito. Essas lesões ainda mostraram macrófagos formando um granuloma desorganizado, corpúsculos residuais de amastigotas no interior de vacúolos parasitórofos de macrófagos e células intensamente vacuolizadas, sugerindo morte por autofagia. Morte celular por apoptose também pode ser evidenciada através da visualização de outras células apresentando cromatina altame

Characterization of leishmania parasites from archived geimsa-stained slides using nested polymerase chain reaction

In Iran, the clinical presentation of cutaneous leishmaniasis is mainly in the form of dry type [urban form] or wet type [rural form]. The microscopic finding of amastigotes in Giemsa-stained smears is the most practical laboratory test for the diagnosis of cutaneous leishmaniasis. However, determination of parasite species is not possible when using this method. Parasite characterization is made by various biochemical, immunological and molecular methods based on massive culture of the parasites. In this study nested PCR was used both for diagnosis as well as species identification. Giemsa-stained slides from forty-nine patients, that had been included in a drug resistance survey, were used in this study. From the available slides, forty-seven were diagnosed as having leishmaniasis using the nested PCR technique. Twenty of these were Leishmania tropica [L. tropica] and the remaining were Leishmania major [L. major]. Amastigotes were recovered from twenty-nine of these patients after standard treatment. This study revealed that clinically drug-resistant cases are more likely to be infected with L. tropica than with L. major, although this difference was not statistically significant. L. tropica was mostly present in facial lesions while L. major was mostly detected in hand and foot lesions. In patients with more than two lesions, L major was the predominant cause. L tropica was the cause of a more prolonged duration of disease. None of the above findings were, however, statistically significant. It can be concluded that nested PCR is a useful technique for studying the molecular epidemiology of leishmaniasis in the field

Ultrastructure study of experimental cutaneous leishmaniasis before and after dapsone intake

Leishmania strain was isolated from a human case of cutaneous leishmaniasis and inoculated into experimental animals. Group of infected animals were given dapsone at a dose of 25 mg/kg for 3 weeks. Results showed that infected animals suffered from autoamputation of the inoculated foot pad. On the other hand, those receiving dapsone showed complete clinical cure. Transmission electron microscope of both groups revealed the fine structure of Leishmania amastigotes. Those given dapsone showed considerable reversible changes, which did not affect parasitic virulence. The latter has been demonstrated by their ability to infect experimental animals leading to pathological lesion. It should be pointed out that patients treated with this drug and showed marked clinical response must be very carefully examined for fear of residual parasites, which may be the cause of relapse later on

La respuesta de Shock térmico y el dimorfismo de Leishmania / Termic shock response and Leishmania dimorphism

Los protozoos del género Leishmania son parásitos intracelulares con dos formas distintas de desarrollo, promastigote y amastigote, las cuales se presentan durante su ciclo de vida entre el hospedero invertebrado y el hospedero mamífero vertebrado, respectivamente. Durante este ciclo el parásito experimenta grandes variaciones de temperatura y el éxito de la transformación entre una y otra forma es crucial para su supervivencia y reproducción. La respuesta de los organismos a la temperatura o respuesta de shock térmico ha sido bien estudiada en Leishmania, obteniendo promastigores a 26§C y amastigotes a 35§C y demostrando su reconversión al aumentar o disminuir la tempertura. Estos cambios en la morfología están acompañados de cambios bioquímicos y moleculares, con diferencias en las actividades enzimáticas y en la síntesis proteica, la cual incrementa la producción de proteínas de shock térmico, específicamente HSP71 y HSP83, las cuales permanecen constitutivamente expresadas en los amastigotes de Leishmania como una forma de adaptación al medio ambiente del hospedero vertebrado. A nivel molecular se ha demostrado que la mayoría de los genes estructurales de estas proteínas están dispuestos en repeticiones en serie los cuales se transcriben en RNA policistrónicos y son posteriormente procesados por poliadenilación y transplicing; sinembargo no ha sido posible

Use of molecular probes and PCR for detection and typing of Leishmania - a mini-review

The use of molecular tools to detect and type Leishmania species in humans, reservoirs or sandflies has been pursued using different approaches. The polymerase chain reaction provided sensitivity to case this task, since the use of hybridization procedures alone employing specifics probes is hampered due to the low detection limit. In this report, we describe the different molecular targets used in our laboratory, aiming at the detection and specific typing of these protozoa. Different kits based on hybridization assays and PCR amplification using kinetoplast and nuclear targets are described and the results obtained from their use are reported.

Immunogold labeling and cerium cytochemistry of the enzyme ecto-5'-nucleotidase in promastigote forms of Leishmania species

We have applied both enzyme cytochemistry and immunological labeling techniques to characterize the enzyme 5'-nucleotidase (5'-Nase), at the ultrastructural level, in promastigote forms of four Leishmania species: Leishmania amazonensis, Leishmania mexicana, Leishmania donovani and Leishmania chagasi. The cerium phosphate staining was localized at the surface of the cell body, the flagellum and the flagellar pocket membranes of all the parasites studied. The immunogold labelling technique confirmed these results. In this report we localized 5'-Nase in L. chagasi and L. amazonensis which have been implicated respectively in visceral and cutaneous forms of leishmaniasis. In addition, we confirmed the localization of this phosphomonoesterase in the other two species studied. The superior quality of the images, obtained with both methodologies, confirms that these parasites possess mechanisms capable of hydrolyzing nucleotide monophosphates, and that the expression of 5'-Nase is associated with the outer surface of the plasma membrane