RESUMEN
Polyamine synthesis from l-ornithine is essential for Leishmania growth. We have investigated the dependence of Leishmania infection on arginase, which generates l-ornithine, in macrophages from BALB/c, C57BL/6, and nitric oxide synthase II (NOS II)-deficient mouse strains. We have found that N(omega)-hydroxy-l-arginine (LOHA), a physiological inhibitor of arginase, controls cellular infection and also specifically inhibits arginase activity from Leishmania major and Leishmania infantum parasites. The effect was proportional to the course of infection, concentration dependent up to 100 microM, and achieved without an increase in nitrite levels of culture supernatants. Moreover, when the l-arginine metabolism of macrophages is diverted towards ornithine generation by interleukin 4-induced arginase I, parasite growth is promoted. This effect can be reversed by LOHA. Inhibition of NOS II by N(G)-methyl-l-arginine (LNMMA) restores the killing obtained in the presence of interferon (IFN)-gamma plus lipolysaccharide (LPS), whereas the nitric oxide scavenger 2-(4-carboxyphenyl)-4,4,5,5,-tetramethylimidazoline-3-oxide-1-oxyl (carboxy-PTIO) was without effect. However, exogenous l-ornithine almost completely inhibits parasite killing when added in the presence of LOHA to macrophages from NOS II-deficient mice or to BALB/c-infected cells activated with IFN-gamma plus LPS. These results suggest that LOHA is an effector molecule involved in the control of Leishmania infection. In addition, macrophage arginase I induction by T helper cell type 2 cytokines could be a mechanism used by parasites to spread inside the host.
Asunto(s)
Arginasa/antagonistas & inhibidores , Arginina/análogos & derivados , Arginina/farmacología , Inhibidores Enzimáticos/farmacología , Leishmania infantum/efectos de los fármacos , Leishmania major/efectos de los fármacos , Macrófagos/enzimología , Animales , Arginasa/metabolismo , Benzoatos/farmacología , Células Cultivadas , Imidazoles/farmacología , Leishmania infantum/crecimiento & desarrollo , Leishmania major/crecimiento & desarrollo , Macrófagos/parasitología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Ornitina/farmacología , Superóxido Dismutasa/metabolismoRESUMEN
In this report an experimental model of Leishmania infantum (L. infantum) infection in dogs is described. The data presented are derived from an overall and comparative analysis of the clinical outcomes of three groups of dogs intravenously infected with 500,000 promastigotes on different dates (2003, 2006 and 2008). The parasites used for challenge were isolated from a dog having a patent form of leishmaniosis, classified as MCAN/ES/1996/BCN150 zymodeme MON-1. Late-log-phase promastigote forms derived from cultured amastigotes obtained from the spleen of the heavily infected hamsters were used for infection. Only one single infective dose was administered to each dog. After challenge, the animals were monitored for 12 months. To analyze the disease outcome, several biopathological, immunological and parasitological end-points were considered. The analysis of the infected dogs indicated that the development of the clinical disease was very similar in the three experimental challenges, as shown by the immune response, the parasite load and the clinical and histopathological lesions detected at necropsy. A high similarity was also observed between the disease development after the experimental challenge and the one reported to occur in endemic natural infection areas, as various degrees of susceptibility to the disease and even resistance were observed in the experimentally infected animals. We believe that this challenge model faithfully reproduces and mimics the course of a natural infection and that it could be used as a suitable tool for analyzing the efficacy of anti-Leishmania drugs and vaccines.
Asunto(s)
Enfermedades de los Perros/parasitología , Leishmania infantum/crecimiento & desarrollo , Leishmaniasis Visceral/veterinaria , Animales , Anticuerpos Antiprotozoarios/biosíntesis , Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/inmunología , Cricetinae , Enfermedades de los Perros/inmunología , Perros , Ensayo de Inmunoadsorción Enzimática/veterinaria , Femenino , Riñón/parasitología , Riñón/patología , Leishmania infantum/inmunología , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/parasitología , Hígado/parasitología , Hígado/patología , Ganglios Linfáticos/parasitología , Masculino , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Bazo/parasitología , Bazo/patologíaRESUMEN
The protective potential against Leishmania infection of the Leishmania chimerical Q protein administered as a single (Q) or double dose (Q+Q) without adjuvant was analyzed in a double-blind placebo controlled experiment in dogs. During vaccination the protein induced an intense early anti-Q response but no reactivity against total Leishmania infantum proteins was detected. Several end-points were taken into consideration. In the vaccinated animals the amount and intensity of clinical symptoms was lower than in the control group. Pathological signs of disease were observed in liver, kidney and spleen of all dogs from the control group in contrast to the normal appearance of the organs of the vaccinated animals. Vaccination was able to induce parasite clearance in most dogs. Only 1/7 dog was parasite DNA positive in skin in the Q group in contrast to 6/7 dogs in control and 4/7 in Q+Q. Significant anti-SLA clearance was observed in the vaccinated animals at the end of the study. Differences between control and vaccinated animals were also observed at the biochemical level, DTH and nitrite oxide production.
Asunto(s)
Enfermedades de los Perros/prevención & control , Leishmania infantum/inmunología , Vacunas contra la Leishmaniasis/inmunología , Leishmaniasis Visceral/veterinaria , Proteínas Protozoarias/inmunología , Animales , Anticuerpos Antiprotozoarios/sangre , Enfermedades de los Perros/inmunología , Enfermedades de los Perros/patología , Perros/parasitología , Método Doble Ciego , Femenino , Inmunoglobulina G/sangre , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/patología , Leishmaniasis Visceral/prevención & control , MasculinoRESUMEN
It has been reported that the level of protection provided by vaccines against murine visceral leishmaniasis (VL) is low and that progress in research on VL may be due to the lack of appropriate models to study protective immunity. We have analysed the immunohistological features occurring in BALB/c mice after intravenous administration of 10(3), 10(5) and 10(6) parasites of Leishmania infantum. Our results show that in all cases parasite administration leads to the establishment of infection and to the development of quantifiable immunohistological features which are dependent on the inoculum size. This study demonstrates that differences in the parasite challenge result in changes in the evolution of some of the parameters associated with the degree of the infection in the BALB/c model: level of anti-Leishmania antibodies, up-regulation of spleen arginase activity, balance between IFN-gamma and IL-10, extent of lymphoid follicle depletion in the splenic white pulp and ineffective development of hepatic granulomas. Also, and depending on the initial infectious inoculum, the absence of parasites in the bone marrow and the number of mature and empty type granulomas were parameters associated with protection. We think that in this model a challenge of the order of 10(5) parasites should prove useful for vaccine studies against VL.