RESUMO
OBJECTIVE: To investigate the in vitro activity, synergism, cytotoxicity and cellular immunological response, as well as the molecular affinity between amphotericin B (AmB) and crotamine (CTA), derived from Crotalus durissus terrificus venom against Leishmania amazonensis. METHODS: This study performed the inhibition of promastigotes and amastigotes' growth under different concentrations of the drug and pharmacological combinations (AmB + CTA) based on the Berimbaum method (synergism study). The lactate dehydrogenase (LDH) quantification method was used to determine the cytotoxicity of the drug and combinations employing four cell lines (J774, HepG2, VERO, and C2C12). Following, the levels of Tumour Necrose Factor-alpha (TNF-α) and Interleukin-12 (IL-12) cytokines, using enzyme-linked immunosorbent assay (ELISA) and nitrites, as an indirect measure of Nitric Oxide (NO), using the Griess reaction were assessed in the supernatants of infected macrophages. In silico approach (molecular docking and dynamics) and binding affinity (surface plasmon resonance) between the drug and toxin were also investigated. RESULTS: CTA enhanced AmB effect against promastigote and amastigote forms of L. amazonensis, decreased the drug toxicity in different cell lines and induced the production of important Th1-like cytokines and NO by infected macrophages. The pharmacological combination also displayed consistent molecular interactions with low energy of coupling and a concentration-dependent profile. CONCLUSION: Our data suggest that this pharmacological approach is a promising alternative treatment against L. amazonensis infection due to the improved activity (synergistic effect) achieved against the parasites' forms and to the decreased cytotoxic effect.
Assuntos
Antiprotozoários , Venenos de Crotalídeos , Anfotericina B/metabolismo , Anfotericina B/toxicidade , Animais , Antiprotozoários/farmacologia , Venenos de Crotalídeos/química , Crotalus/metabolismo , Citocinas/metabolismo , Simulação de Acoplamento Molecular , Óxido Nítrico/metabolismoRESUMO
This study aimed to evaluate the in vivo anti-Leishmania amazonensis activity of a Phospholipase A2 (Asp49-PLA2), isolated from Bothrops jararacussu venom, encapsulated in liposomes as a modified toxin release system. The activity of the liposomes was evaluated in BALB/c mice, previously infected with 1×105 of the parasite's promastigotes. The size of the paw lesion in Asp49-PLA2-liposomal-treated animals, after 21days, was observed as decreasing by 16% relative to the untreated control group and 12% by the Glucantime®-treated animals, which was used as a reference drug. At the end of the treatment, the animals were sacrificed and the paw and lymph node tissues were collected. Part of the collection was used to recover amastigotes and another to quantify cytokines and nitrites. In the group treated with Asp49-PLA2-liposomes the parasitic load was observed to be reduced by 73.5% in the macerated lymph node, compared to the control group. Comparatively, in the paw tissue was observed a reduction of 57.1%. The infected groups treated with Asp49-PLA2-liposomes showed significant production in TNF-α measured in lymph nodes and paw (43.73pg/mL±2.25 and 81.03pg/mL±5.52, respectively) and nitrite levels (31.28µM±0.58 and 35.64µM±5.08) also measured in lymph nodes and paw tissues, respectively, compared to untreated groups. These results indicate that the Asp49-PLA2-loaded liposomes were able to activate the production of some cellular components of the protective TH1 response during the infection, constituting a promising tool for inducing the microbicidal activity of the Leishmania-infected macrophages.
Assuntos
Venenos de Crotalídeos/metabolismo , Leishmania/fisiologia , Leishmaniose Cutânea/terapia , Lipossomos/metabolismo , Linfonodos/imunologia , Macrófagos/imunologia , Fosfolipases A2/metabolismo , Proteínas de Répteis/metabolismo , Animais , Anti-Infecciosos/metabolismo , Bothrops , Modelos Animais de Doenças , Humanos , Lipossomos/uso terapêutico , Linfonodos/parasitologia , Macrófagos/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Nitritos/metabolismo , Carga Parasitária , Fosfolipases A2/uso terapêutico , Proteínas de Répteis/uso terapêutico , Células Th1/imunologia , Terapias em Estudo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
American tegumentary leishmaniasis (ATL) is considered a neglected disease, for which an effective vaccine or an efficient diagnosis is not yet available and whose chemotherapeutic arsenal is threatened by the emergence of resistance by etiological agents such as Leishmania amazonensis. ATL is endemic in poor countries and has a high incidence in Brazil. Vaccines developed from native parasite fractions have led to the identification of defined antigenic subunits and the development of vaccine adjuvant technology. The purpose of the present study was to develop and compare preparations based on membrane antigens from L. amazonensis, as a biotechnological prototype for the immunoprophylaxis of the disease in a murine experimental model. For this purpose, batches of biodegradable polymeric micro/nanoparticles were produced, characterized and compared with other parasite's antigens in solution. All preparations containing membrane antigens presented low toxicity on murine macrophages. The in vivo evaluation of immunization efficacy was performed against a challenge with L. amazonensis, along with an evaluation of the immune response profile generated in BALB/C mice. The animals were followed for sample processing and quantification of serum-specific cytokines, nitrites and antibodies. The sera of animals immunized with the non-encapsulated antigen formulations showed higher intensities of nitrites and total IgGs. This approach evidenced the importance of the biological studies involving the immune response of the host against the parasite being interconnected and related to the subfractionation of its proteins in the search for more effective vaccine candidates.
Assuntos
Antígenos de Protozoários/imunologia , Leishmania/imunologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Cutânea/imunologia , Leishmaniose/imunologia , Macrófagos/imunologia , Proteínas de Membrana/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Células Cultivadas , Citocinas/sangue , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , Nanopartículas , Óxido Nítrico/metabolismoAssuntos
Bothrops/metabolismo , Venenos de Crotalídeos/metabolismo , Leishmania/fisiologia , Leishmaniose Cutânea/tratamento farmacológico , Lipossomos/farmacologia , Macrófagos/efeitos dos fármacos , Fosfolipases A2/farmacologia , Animais , Linhagem Celular , Sobrevivência Celular , Sistemas de Liberação de Medicamentos , Lipossomos/metabolismo , Macrófagos/parasitologia , Macrófagos/fisiologia , Camundongos , Nitritos/metabolismo , Fosfolipases A2/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND/AIMS: To evaluate antileishmanial activity of crotamine, a toxin isolated from Crotalus durissus terrificus, in solution form and encapsulated in biodegradable microparticles in vitro. METHODS: Particles were analyzed on-chip by surface plasmon resonance and characterized by testing their diameters, zeta potential and encapsulation rate. The viability of promastigotes as well as murine macrophages was assessed. Furthermore, the phagocytic index was determined for macrophages, and cell supernatants were collected for the determination of TNF-α levels. An infection assay using Leishmania amazonensis-infected macrophages was also conducted. RESULTS: The diameters and zeta potential of control particles (1.35 µm; -12.3 mV) and of those containing crotamine (3.09 µm; -20.9 mV) were adequate for the assays conducted. Crotamine-loaded particles were better captured by macrophages than control particles (increase of 12% in the phagocytic index), leading to increased TNF-α levels (196 pg/ml), and they also induced a significant decrease in the numbers of amastigotes compared to infected macrophages only. CONCLUSION: The approach presented here opens the possibility of working with safe concentrations of encapsulated toxins to reach antileishmanial effects.