Hepatic fibropoiesis in dogs naturally infected with Leishmania (Leishmania) infantum treated with liposome-encapsulated meglumine antimoniate and allopurinol.

Hepatic fibropoiesis in canine visceral leishmaniasis (CVL) were evaluated by histological (morphometrical collagen deposition) and immunohistochemical assays characterizing alpha-actin (α-SMA), vimentin, calprotectin (L1 antigen), and TGF-ß in 46 naturally infected dogs with Leishmania infantum treated with liposome-encapsulated meglumine antimoniate and allopurinol separately and in combination. Six treatment groups were defined: meglumine antimoniate encapsulated in nanometric liposomes (LMA), allopurinol (ALLOP); liposome-encapsulated meglumine antomoniate combined with allopurinol (LMA+ALLOP); empty liposomes (LEMP); empty liposomes combined with allopurinol (LEMP+ALLOP) and saline. Relative liver weight was lower in LMA, LMA+ALLOP, and ALLOP groups compared to the LEMP control. Significantly lower granulomatous chronic inflammatory reaction was seen in the ALLOP group compared to a control group. Calprotectin was lowest in liver of those dogs showing lower numbers of intralobular hepatic granulomas. Collagen deposits were significantly higher in LMA compared to ALLOP, LEMP+ALLOP, and Saline groups. LMA+ALLOP group collagen deposition was higher than dogs treated only with allopurinol. Immunohistochemical analysis showed significant higher α-SMA in hepatic stellate cells (HSCs), hepatic perisinusoidal cells, in control groups than LMA+ALLOP and LEMP+ALLOP. Alpha-actin and Vimentin positive cells were diffusely distributed throughout the liver parenchyma in the hepatic lobule, mainly in HSCs. Vimentin expression was significantly higher in the saline group than in the ALLOP group. Our data suggest that allopurinol inhibits HSC and results in lower collagen deposits in liver during CVL progression, as supported by the significantly lower expression of TGF-ß in the ALLOP group compared to other groups. Results demonstrated that treatment with allopurinol inhibited chronic granulomatous inflammatory reaction and hepatic fibrosis in CVL.

Humoral immunological profile and parasitological statuses of Leishmune vaccinated and visceral leishmaniasis infected dogs from an endemic area.

Dogs represent the major reservoir of Leishmaniao chagasi and vaccination against the canine disease is a potential control strategy. However, seroconversion occurs post-vaccine and hence, there is need to discriminate between the former group and naturally infected dogs. The present study represents a comparison of the humoral immunological profiles of both groups using Leishmania soluble antigen (LSA) and fucose-mannose ligand (FML). For both categories, ear skin samples were evaluated immunohistochemically and through PCR, that was also performed in blood specimens, as well as their ability to infect Lutzomyia (Lutzomyia) longipalpis. All these tests showed negative results for the vaccinated dogs. Differences between groups were observed regarding IgG, IgG2 and IgE absorbances as determined by FML-ELISA, and for IgG1 and IgE absorbances as measured by LSA-ELISA, showing that Leishmune-immunised animals and VL naturally infected dogs present different immunological profiles, even though these differences cannot be used to distinguish between these two groups of dogs.

Cervical, mandibular, and parotid lymph nodes of dogs naturally infected with Leishmania infantum: a histopathologic and immunohistochemistry study and its correlation with facial skin lesions.

The parasite load in cervical, mandibular, and parotid lymph nodes and in the skin of the nose and the pinna from dogs infected with Leishmania infantum were investigated by histologic and immunohistochemical studies. Twenty-two infected dogs with and without signs of infection were examined to demonstrate correlation of signs with parasite load and the correlation of facial skin lesions with parasites in regional lymph nodes. Chronic inflammation of the skin was demonstrated in infected dogs that had no gross skin lesions, confirming that normal-appearing skin can harbor the parasite, likely playing a role in transmission. Dogs with facial skin lesions showed a higher parasite load in parotid lymph nodes than dogs without lesions of the facial skin, based on Leishman-Donovan unit analysis. Based on immunohistochemical analysis, parasite load in parotid and cervical nodes was correlated with that of skin of the nose and pinna, as was the parasite load in mandibular lymph nodes and skin of the external nose. We demonstrated a logical involvement of the lymphatic vessels and their specific anatomic draining sites.

Hepatic granulomas in canine visceral leishmaniasis and clinical status

The histopathological description of intralobular hepatic granulomas in animals with a defined clinical status (asymptomatic, oligosymptomatic and symptomatic animals) was reported. Seventy-one mongrel dogs naturally infected with Leishmania chagasi were obtained from two Brazilian endemic areas: João Pessoa, PB and Belo Horizonte, MG. The hepatic parasite load was determined and compared to granuloma formation. Liver fragments from all infected animals showed remarkable leishmaniotic granulomatous inflammatory reaction. Granulomas with variable size were constituted by macrophages (parasitized or not with amastigotes of L. chagasi), some epithelioid cells, small numbers of lymphocytes, plasma cells, and rare neutrophils. Asymptomatic dogs had higher numbers of granulomas than oligosymptomatic and symptomatic animals from both geographical regions. However, the average diametric size of granulomas was very heterogeneous in all groups, independently of the geographic region (P>0.05). Parasite tissue load did not show any difference among liver fragments of all animals, especially when considering the defined clinical status and/or their geographic origin

Descreve-se a formação de granulomas hepáticos na leishmaniose canina em animais com classificação clínica definida - assintomáticos, oligossintomáticos e sintomáticos. Setenta e um animais, sem raça definida e naturalmente infectados com Leishmania chagasi, foram obtidos de duas regiões endêmicas brasileiras: João Pessoa, PB e Belo Horizonte, MG. A carga parasitária tecidual foi determinada mediante emprego do Leishmania Donovani Units (LDU) e comparada com a formação de granulomas hepáticos. Fragmentos de fígado de todos os animais infectados mostraram reação granulomatosa notadamente leishmaniótica. Granulomas de variáveis tamanhos eram constituídos por macrófagos, parasitados ou não com formas amastigotas de L. chagasi, algumas células epitelióides, pequeno número de linfócitos e plasmócitos, e raros neutrófilos. Cães assintomáticos apresentaram maior número de granulomas do que os animais oligossintomáticos e sintomáticos, em ambas as regiões geográficas. As médias dos diâmetros foram heterogêneas em todos os grupos, independente da região geográfica (P>0,05). Quanto ao parasitismo (LDU), não houve diferença entre as amostras de fígado, especialmente quando se consideraram a classificação clínica e a região geográfica

Histopathological and immunohistochemical study of type 3 complement receptors (CD11b/CD18) in livers and spleens of asymptomatic and symptomatic dogs naturally infected with Leishmania (Leishmania) chagasi.

Leishmania promastigotes interact with macrophages through the association of multiple membrane surface receptors. Macrophage complement receptor CR3 (CD11b/CD18 or Mac-1) has been implicated in the interaction of both human and murine macrophages with serum-opsonized promastigotes. The aim of this study was to determine CR3 expression in the livers and spleens of dogs naturally infected with Leishmania (Leishmania) chagasi. CR3 expression in liver was higher in asymptomatic than in symptomatic animals. Moreover, the hepatic parasitism load determined by immunocytochemical analysis was lower in parallel with higher numbers of granulomas. In contrast, in spleens, CR3 expression was higher in symptomatic animals than in asymptomatic ones. However, the tissue parasite load was greater in spleens of symptomatic dogs. There was a strict correlation between the parasite load and cellular CR3 expression in the spleens of dogs naturally infected with L. chagasi. CR3 macrophage integrins could be essential receptors for Leishmania survival. Considering that the symptomatic animals showed higher parasite loads and higher CD11b/CD18 expression in their spleens, we can conclude that these splenic cells (monocyte-macrophages) might serve to perpetuate intracellular infection.

Pharmacokinetic and parasitological evaluation of the bone marrow of dogs with visceral leishmaniasis submitted to multiple dose treatment with liposome-encapsulated meglumine antimoniate

The aim of the present study was to evaluate the impact of a multiple dose regimen of a liposomal formulation of meglumine antimoniate (LMA) on the pharmacokinetics of antimony in the bone marrow of dogs with visceral leishmaniasis and on the ability of LMA to eliminate parasites from this tissue. Dogs naturally infected with Leishmania chagasi received 4 intravenous doses of either LMA (6.5 mg antimony/kg body weight, N = 9), or empty liposomes (at the same lipid dose as LMA, N = 9) at 4-day intervals. A third group of animals was untreated (N = 8). Before each administration and at different times after treatment, bone marrow was obtained and analyzed for antimony level (LMA group) by electrothermal atomic absorption spectrometry, and for the presence of Leishmania parasites (all groups). There was a significant increase of antimony concentration from 0.76 æg/kg wet organ (4 days after the first dose) to 2.07 æg/kg (4 days after the fourth dose) and a half-life of 4 days for antimony elimination from the bone marrow. Treatment with LMA significantly reduced the number of dogs positive for parasites (with at least one amastigote per 1000 host cells) compared to controls (positive dogs 30 days after treatment: 0 of 9 in the LMA group, 3 of 9 in the group treated with empty liposomes and 3 of 8 in the untreated group). However, complete elimination of parasites was not achieved. In conclusion, the present study showed that multiple dose treatment with LMA was effective in improving antimony levels in the bone marrow of dogs with visceral leishmaniasis and in reducing the number of positive animals, even though it was not sufficient to achieve complete elimination of parasites.

Pharmacokinetic and parasitological evaluation of the bone marrow of dogs with visceral leishmaniasis submitted to multiple dose treatment with liposome-encapsulated meglumine antimoniate.

The aim of the present study was to evaluate the impact of a multiple dose regimen of a liposomal formulation of meglumine antimoniate (LMA) on the pharmacokinetics of antimony in the bone marrow of dogs with visceral leishmaniasis and on the ability of LMA to eliminate parasites from this tissue. Dogs naturally infected with Leishmania chagasi received 4 intravenous doses of either LMA (6.5 mg antimony/kg body weight, N = 9), or empty liposomes (at the same lipid dose as LMA, N = 9) at 4-day intervals. A third group of animals was untreated (N = 8). Before each administration and at different times after treatment, bone marrow was obtained and analyzed for antimony level (LMA group) by electrothermal atomic absorption spectrometry, and for the presence of Leishmania parasites (all groups). There was a significant increase of antimony concentration from 0.76 microg/kg wet organ (4 days after the first dose) to 2.07 microg/kg (4 days after the fourth dose) and a half-life of 4 days for antimony elimination from the bone marrow. Treatment with LMA significantly reduced the number of dogs positive for parasites (with at least one amastigote per 1000 host cells) compared to controls (positive dogs 30 days after treatment: 0 of 9 in the LMA group, 3 of 9 in the group treated with empty liposomes and 3 of 8 in the untreated group). However, complete elimination of parasites was not achieved. In conclusion, the present study showed that multiple dose treatment with LMA was effective in improving antimony levels in the bone marrow of dogs with visceral leishmaniasis and in reducing the number of positive animals, even though it was not sufficient to achieve complete elimination of parasites.

Distribution of liposome-encapsulated antimony in dogs.

The achievement of complete cure in dogs with visceral leishmaniasis is currently a great challenge, since dogs are the main reservoir for the transmission of visceral leishmaniasis to humans and they respond poorly to conventional treatment with pentavalent antimonials. In order to improve the efficacy of treatment, we developed a novel formulation for meglumine antimoniate based on the encapsulation of this drug in freeze-dried liposomes (LMA). The aim of the present study was to evaluate the biodistribution of antimony (Sb) in dogs following a single intravenous bolus injection of LMA. Four healthy male mongrel dogs received LMA at 3.8 mg Sb/kg body weight and were sacrificed 3, 48 and 96 h and 7 days later. Antimony was determined in the blood, liver, spleen and bone marrow. In the bone marrow, the highest Sb concentration was observed at 3 h (2.8 microg/g wet weight) whereas in the liver and spleen it was demonstrated at 48 h (43.6 and 102.4 microg/g, respectively). In these organs, Sb concentrations decreased gradually and reached levels of 19.1 microg/g (liver), 28.1 microg/g (spleen) and 0.2 microg/g (bone marrow) after 7 days. Our data suggest that the critical organ for the treatment with LMA could be the bone marrow, since it has low Sb levels and, presumably, high rates of Sb elimination. A multiple dose treatment with LMA seems to be necessary for complete elimination of parasites from bone marrow in dogs with visceral leishmaniasis.

Distribution of liposome-encapsulated antimony in dogs

The achievement of complete cure in dogs with visceral leishmaniasis is currently a great challenge, since dogs are the main reservoir for the transmission of visceral leishmaniasis to humans and they respond poorly to conventional treatment with pentavalent antimonials. In order to improve the efficacy of treatment, we developed a novel formulation for meglumine antimoniate based on the encapsulation of this drug in freeze-dried liposomes (LMA). The aim of the present study was to evaluate the biodistribution of antimony (Sb) in dogs following a single intravenous bolus injection of LMA. Four healthy male mongrel dogs received LMA at 3.8 mg Sb/kg body weight and were sacrificed 3, 48 and 96 h and 7 days later. Antimony was determined in the blood, liver, spleen and bone marrow. In the bone marrow, the highest Sb concentration was observed at 3 h (2.8 æg/g wet weight) whereas in the liver and spleen it was demonstrated at 48 h (43.6 and 102.4 æg/g, respectively). In these organs, Sb concentrations decreased gradually and reached levels of 19.1 æg/g (liver), 28.1 æg/g (spleen) and 0.2 æg/g (bone marrow) after 7 days. Our data suggest that the critical organ for the treatment with LMA could be the bone marrow, since it has low Sb levels and, presumably, high rates of Sb elimination. A multiple dose treatment with LMA seems to be necessary for complete elimination of parasites from bone marrow in dogs with visceral leishmaniasis

Novel methods for the encapsulation of meglumine antimoniate into liposomes

The antimonial drug, meglumine antimoniate, was successfully encapsulated in dehydration-rehydration vesicles and in freeze-dried empty liposomes (FDELs). High encapsulation efficiencies (from 28 to 58 percent) and low weight ratios of lipids to encapsulated antimony (from 1:0.15 to 1:0.3) were achieved. These formulations, contrary to those obtained by conventional methods, can be stored as intermediate lyophilized forms and reconstituted just before use. The efficacy of FDEL-encapsulated meglumine antimoniate was evaluated in hamsters experimentally infected with Leishmania chagasi. A significant reduction of liver parasite burdens was observed in animals treated with this preparation, when compared to control animals treated with empty liposomes. In contrast, free meglumine antimoniate was found to be inefficient when administered at a comparable dose of antimony. This novel liposome-based meglumine antimoniate formulation appears to be promising as a pharmaceutical product for the treatment of visceral leishmaniasis.

Leishmaniose visceral canina: estudo imunocitoquímico dos antígenos do MHC classe II no fígado e órgäos linfóides / Canine visceral leishmaniasis: immunocytochemical study of the MHC class II antigens in liver and lymphoid organs

O objetivo deste trabalho foi o de estudar a expressäo imunocitoquímica dos antígenos do MHC classe II no fígado e órgäos linfóides (baço, linfonodos e placas de Peyer) de cäes, sem raça definida, experimentalmente e naturalmente infectados com Leishmania chagasi. Cortes em criostato de fígado, baço, linfonodos e placas de Peyer foram corados pela técnica imunocitoquímica peroxidase anti-peroxidase (PAP). A marcaçäo imunocitoquímica para os antígenos do MHC classe II revelou a mesma topografia em todos os órgäos examinados de todos os animais infectados. Entretanto, a expressäo dos antígenos do MHC classe II foi menos intensa nos linfonodos cervicais e abdominais dos cäes naturalmente infectados, indicando que L. chagasi é capaz de inibir a expressäo dos antígenos do MHC classe II

Comparaçäo entre o teste imunoenzimático e a reaçäo de imunofluorescência indireta no diagnóstico sorológico da leishmaniose tegumentar americana canina experimental / Comparison between the enzyme-linked immunossorbent assay and the indirect fluorescent antibody test in the diagnosis of canine experimental tegumentar leishmaniasis

Trinta e um cäes foram inoculados experimentalmente com Leishmania (Viania) braziliensis e acompanhados clínica e laboratorialmente por 539 dias. O teste imunoenzimático (ELISA) e a reaçäo de imunofluorescência indireita (IFI) foram utilizados no acompanhamento da resposta imune humoral e avaliados como métodos de diagnóstico durante a evoluçäo da doença. Os resultados apontam maior sensibilidade do ELISA, indicando ser este método mais adequado que IFI como auxiliar para o diagnóstico sorológico da leishmaniose tegumentar americana canina

Immunocytochemical identification of Leishmania and Trypanosoma cruzi amastigotes in situ with homologous and heterologous polyclonal antibodies

O método da peroxidase-antiperoxidase foi utilizado para estudar as propriedades imunocitoquímicas de Leishmanias e de amastigotas do Trypanosoma cruzi, in situ, após os tecidos terem sido submetidos a diferentes tipos de fixaçäo. Anti-soros foram obtidos de coelhos cronicamente infectados com três cepas de T. cruzi ou imunizados com L. mexicana amazonensis e L. braziliensis guyanensis e aplicados nos cortes histológicos de 5*m de espessura. Os antígenos de T. cruzi foram coroados muito bem pelos três soros anti-T. cruzi e pelos dois soros anti-Leishmania com diluiçöes entre 1:1.000 e 1:2. Diferentemente, os antígenos de Leishmania foram revelados pelos soros anti-Leishmania somente em baixas diluiçöes, ou seja, entre 1:60 e 1:160 enquanto que os soros anti-T. cruzi, mesmo nestas diluiçöes baixas quando usados para revelar Leishmania. Embora näo haja explicaçäo clara para esta reaçäo imunocitoquímicacruzada "reversa-monodirecional" entre Leishmania e amastigotas de T. cruzi os resultados do presente trabalho mostram que anticorpos policlonais contra diferentes espécies de Leishmania, quando usados para detecçäo imunocitoquímica de Leishmania e T. cruzi in situ, reagem mais fortemente com amastigotas de T. cruzi do que com espécies homólogas

The change of behavior of two strains of Leishmania after cultivation in a defined medium

Duas cepas de Leishmania originalmente isoladas in vitro de casos humanos de leishmaniose cutânea e que ab initio näo infectaram animais de laboratório, tornaram-se infectantes para hamnsters após serem mantidos por vários anos em meio de cultura quimicamente definido. Foram realizadas observaçöes sobre o crescimento de promastigotas in vitro, curso da infecçäo em hamsters, morfologia das amastigotas, mobilidade eletroforética de oito enzimas solúveis. Foram obtidas informaçöes sobre a densidade de flutuaçäo do n-DNA e do k-DNA e uma das cepas foi testada contra anticorpos monoclonais. Ambas as cepas permanecem sem identificaçäo precisa