Análise imunológica e anátomo-patológica de cães naturalmente infectados com Leishmania (Leishmania) chagasi tratados com antimoniato de meglumina encapsulado em lipossomas nanométricos e alopurinol

Abstract

Leishmania are intracellular obligate parasites that reside predominantly in mononuclear phagocyte cells of vertebrate hosts. In the Americas, dogs (Canis lupus familiaris) are considered the main domestic reservoirs for human visceral leishmaniasis (HVL). In Brazil, the measures proposed for the LV control, consider early diagnosis and treatment of human cases, residual spraying insecticides to combat the insect vector and dogs serological survey, with following euthanasia of positive animals. However, dogs euthanasia is not sustained by economic, social and ethical concerns. The treatment of infected dog with conventional therapy is questionable thus use of liposomal formulations with leishmanicidal activity is a promising strategy. Added to this, the co-treatment with leishmaniostatic drugs represents a good strategy in canine visceral leishmaniasis (CVL) treatment. In our study, treated dogs were divided in six groups as follow: Group I composed by eight dogs treated with meglumine antimoniate encapsulated in liposomes (LMA) associated with allopurinol (Allop) denominated LMA+Allop; Group II composed by eight dogs treated with LMA; Group III composed by eight dogs treated with allopurinol (Allop); Group IV composed by eight dogs treated with empty liposome (LEMP) associated with Allop denominated group LEMP+Allop; Group V composed by eight dogs treated with LEMP. Group VI composed by twelve dogs treated with saline solution (SG). The recommended therapeutic protocol consisted in six administrations every 96h, of 6.5 mg/kg/i.v. LMA; six administrations every 96h, intravenously, into the corresponding volume of empty liposome; and 20mg/kg of allopurinol, once daily for a period of 140 days. Clinical assessments were done before treatment begins, named time (T0) and at moments corresponding to 50, 80, 110, 140, 170 and 201 days after establishment of treatment (T50, T80, T110, T140, T170 and T201). Similarly, peripheral blood samples were collected at time T0, T60, T140 and T200. The procedure was peripheral blood mononuclear immunostaining cells with anti-CD11b, anti-MHCII, anti-TL-2, anti-CD4 and anti-CD8 evaluated by the flow cytometry technique. Skin biopsies, of the inside region of the right pina, were collected at T0, T60, T140. In T200 samples of skin, liver, spleen, superficial cervical lymph nodes were collected during necropsy, which was processed for routine histological evaluations. Semi-quantitative and qualitative histologic studies and parasitism evaluation (immunohistochemistry) were performed using conventional optical microscopy. The Allop group concerning mainly associated with LMA promoted significantly reduced parasitis load (skin, liver and spleen) and clinical improvement of all animals. However, despite this improvement, in dogs studied, was not observed immune response modulation (CD4/CD8 lymphocyte population frequency). Furthermore, the TLR-2 and CR3 expression, in CD14+ monocytes, despite variations observed, along the kinetics of different treatment protocols, did not correlate with the clinical picture. Allopurinol treated dogs mainly associated with LMA showed remission of dermatological classic signs of LVC. Those Allop group, mainly LMA associated showed histological alterations decreased mainly characterized by a lower chronic dermatitis frequency. Allopurinol treated dogs and meglumine antimoniate encapsulated in liposomes treated groups showed significantly lower relative liver weight than in the empty liposomes group. Allopurinol treated dogs with the various associations and treated dogs with encapsulated meglumine antimoniate liposomes when compared to empty liposomes group, showed a decrease of intralobular hepatic granulomas number and hepatic chronic signs. Meglumine antimoniate encapsulated liposomes group associated with allopurinol showed significantly lower in relative weight of the spleen than in the allopurinol-treated group and control. The treatment in the different experimental conditions did not showed significantly alteration in conventional splenic lesions as white pulp T-dependent deplation zone that was observed in most animals studied. The LMA+Allop and Allop groups showed a significant sinnus medullary cellularity reduction as compared to the empty liposome group with a lower trend tissue parasitism. We could say that the animals under different therapeutic protocols, despite the clinical and parasitological improvement, must be accompanied by a longer period of time, since histological changes such as fibrosis and degeneration were mainly found in the liver of LMA + Allop and Allop groups.