RESUMEN
Visceral leishmaniasis (VL) is a neglected tropical disease found in tropical and subtropical regions in the world. The therapeutics used for the treatment against disease presents problems, mainly related to drug toxicity, route of administration, high cost and/or by emergence of resistant strains. In this context, the search for alternative antileishmanial candidates is desirable. Recently, a naphthoquinone derivative namely 2-(2,3,4-tri-O-acetyl-6-deoxy-ß-L-galactopyranosyloxy)-1,4-naphthoquinone or Flau-A showed an effective in vitro biological action against Leishmania infantum. In the present study, the efficacy of this naphthoquinone derivative was evaluated in an in vivo infection model. BALB/c mice (n = 12 per group) were infected and later received saline or were treated with empty micelles (B/Mic), free Flau-A or it incorporated in Poloxamer 407-based micelles (Flau-A/Mic). The products were administered subcutaneously in the infected animals, which were then euthanized one (n = 6 per group) and 15 (n = 6 per group) days post-therapy, when immunological and parasitological evaluations were performed. Results showed that animals treated with Flau-A or Flau-A/Mic produced significantly higher levels of antileishmanial IFN-γ, IL-12, TNF-α, GM-CSF, nitrite and IgG2a isotype antibody, when compared to data found in the control (saline and B/Mic) groups; which showed significantly higher levels of parasite-specific IL-4, IL-10 and IgG1 antibody. In addition, animals receiving free Flau-A or Flau-A/Mic presented also significant reductions in the parasite load in their spleens, livers, bone marrows and draining lymph nodes, when compared to the controls. A low hepatic and renal toxicity was also found. Overall, Flau-A/Mic showed better immunological and parasitological results, when compared to the use of free molecule. In conclusion, preliminary data suggest that this composition could be considered in future studies as promising therapeutic candidate against VL.
Asunto(s)
Antiprotozoarios/química , Antiprotozoarios/uso terapéutico , Leishmania infantum/efectos de los fármacos , Leishmaniasis Visceral/tratamiento farmacológico , Naftoquinonas/química , Naftoquinonas/uso terapéutico , Animales , Antiprotozoarios/farmacología , Femenino , Leishmania infantum/genética , Leishmania infantum/fisiología , Ratones , Ratones Endogámicos BALB C , Micelas , Naftoquinonas/farmacología , Carga de Parásitos , Reacción en Cadena en Tiempo Real de la Polimerasa , Bazo/parasitologíaRESUMEN
Leishmaniasis is a parasitic disease caused by Leishmania protozoa, which presents a large spectrum of clinical manifestations. In the present study, a quinoline derivative salt named N-(2-((7-chloroquinolin-4-yl)amino)ethyl)-N-(prop-2-yn-1-yl)prop-2-yn-1-aminium chloride or QDS3 was in vitro and in vivo tested against L. infantum by means of its incorporation in Poloxamer 407-based polymeric micelles (QDS3/M). The in vitro antileishmanial activity of QDS3 and QDS3/M was investigated in L. infantum promastigotes, axenic amastigotes and infected macrophages. BALB/c mice were infected with L. infantum, and parasitological parameters were evaluated 1 and 15 days post-treatment by determining the parasite load by a limiting dilution assay, besides a quantitative PCR (qPCR) method. Immunological response was assessed based on production of cellular cytokines, as well as by quantification of nitrite levels and specific antibodies. In vitro results showed that QDS3 free or in micelles presented effective antileishmanial action against both parasite stages, being more effective in amastigotes. In vivo data showed that treatment using QDS3 or QDS3/M reduced the parasite load in the livers, spleens, draining lymph nodes (dLN) and bone marrows of the treated animals, 1 and 15 days after treatment, when compared to values found in the control groups. Additionally, treated mice developed a polarized Th1-type immune response, with higher levels of IL-12, IFN-γ, GM-CSF and nitrite, besides high production of specific IgG2a antibodies, when compared to the controls. Parasitological and immunological data obtained using the micellar composition were better than the others. In conclusion, QDS3, mainly when applied in a delivery adjuvant system, could be considered for future studies as therapeutic candidate against VL.
Asunto(s)
Antiprotozoarios , Leishmania infantum , Leishmaniasis Visceral , Leishmaniasis , Quinolinas , Animales , Antiprotozoarios/uso terapéutico , Leishmaniasis/parasitología , Leishmaniasis Visceral/tratamiento farmacológico , Leishmaniasis Visceral/parasitología , Ratones , Ratones Endogámicos BALB C , Micelas , Nitritos/uso terapéutico , Polímeros/uso terapéutico , Quinolinas/uso terapéuticoRESUMEN
The control measures against visceral leishmaniasis (VL) include a precise diagnosis of disease, the treatment of human cases, and reservoir and vector controls. However, these are insufficient to avoid the spread of the disease in specific countries worldwide. As a consequence, prophylactic vaccination could be interesting, although no effective candidate against human disease is available. In the present study, the Leishmania infantum amastin protein was evaluated regarding its immunogenicity and protective efficacy against experimental VL. BALB/c mice immunized with subcutaneous injections of the recombinant protein with or without liposome/saponin (Lip/Sap) as an adjuvant. After immunization, half of the animals per group were euthanized and immunological evaluations were performed, while the others were challenged with L. infantum promastigotes. Forty-five days after infection, the animals were euthanized and parasitological and immunological evaluations were performed. Results showed the development of a Th1-type immune response in rAmastin-Lip and rAmastin-Sap/vaccinated mice, before and after infection, which was based on the production of protein and parasite-specific IFN-γ, IL-12, GM-CSF, and nitrite, as well as the IgG2a isotype antibody. CD4+ T cells were mainly responsible for IFN-γ production in vaccinated mice, which also presented significant reductions in parasitism in their liver, spleen, draining lymph nodes, and bone marrow. In addition, PBMC cultures of treated VL patients and healthy subjects stimulated with rAmastin showed lymphoproliferation and higher IFN-γ production. In conclusion, the present study shows the first case of an L. infantum amastin protein associated with distinct delivery systems inducing protection against L. infantum infection and demonstrates an immunogenic effect of this protein in human cells.
Asunto(s)
Leishmania infantum/inmunología , Leishmaniasis Visceral/inmunología , Proteínas Protozoarias/inmunología , Adyuvantes Inmunológicos/farmacología , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/parasitología , Células Cultivadas , Femenino , Humanos , Inmunidad/inmunología , Interferón gamma/inmunología , Leishmaniasis Visceral/parasitología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/parasitología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/parasitología , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes/inmunología , Células TH1/inmunología , Células TH1/parasitologíaRESUMEN
AIMS: Treatment for visceral leishmaniasis (VL) is hampered by the toxicity and/or high cost of drugs, as well as by emergence of parasite resistance. Therefore, there is an urgent need for new antileishmanial agents. METHODS AND RESULTS: In this study, the antileishmanial activity of a diprenylated flavonoid called 5,7,3,4'-tetrahydroxy-6,8-diprenylisoflavone (CMt) was tested against Leishmania infantum and L amazonensis species. Results showed that CMt presented selectivity index (SI) of 70.0 and 165.0 against L infantum and L amazonensis promastigotes, respectively, and of 181.9 and 397.8 against respective axenic amastigotes. Amphotericin B (AmpB) showed lower SI values of 9.1 and 11.1 against L infantum and L amazonensis promastigotes, respectively, and of 12.5 and 14.3 against amastigotes, respectively. CMt was effective in the treatment of infected macrophages and caused alterations in the parasite mitochondria. L infantum-infected mice treated with miltefosine, CMt alone or incorporated in polymeric micelles (CMt/Mic) presented significant reductions in the parasite load in distinct organs, when compared to the control groups. An antileishmanial Th1-type cellular and humoral immune response were developed one and 15 days after treatment, with CMt/Mic-treated mice presenting a better protective response. CONCLUSION: Our data suggest that CMt/Mic could be evaluated as a chemotherapeutic agent against VL.
Asunto(s)
Antiprotozoarios/administración & dosificación , Leishmaniasis Visceral/tratamiento farmacológico , Animales , Antiprotozoarios/química , Antiprotozoarios/farmacología , Femenino , Flavonoides/administración & dosificación , Flavonoides/química , Flavonoides/farmacología , Inmunidad Celular/efectos de los fármacos , Inmunidad Humoral/efectos de los fármacos , Leishmania infantum/efectos de los fármacos , Leishmania infantum/crecimiento & desarrollo , Leishmania mexicana/efectos de los fármacos , Leishmania mexicana/crecimiento & desarrollo , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/parasitología , Macrófagos/efectos de los fármacos , Macrófagos/parasitología , Ratones , Ratones Endogámicos BALB C , Micelas , Carga de ParásitosRESUMEN
The treatment against visceral leishmaniasis (VL) presents problems, mainly related to the toxicity and/or high cost of the drugs. In this context, a prophylactic vaccination is urgently required. In the present study, a Leishmania protein called LiHyE, which was suggested recently as an antigenic marker for canine and human VL, was evaluated regarding its immunogenicity and protective efficacy in BALB/c mice against Leishmania infantum infection. In addition, the protein was used to stimulate peripheral blood mononuclear cells (PBMCs) from VL patients before and after treatment, as well as from healthy subjects. Vaccination results showed that the recombinant (rLiHyE) protein associated with liposome or saponin induced effective protection in the mice, since significant reductions in the parasite load in spleen, liver, draining lymph nodes, and bone marrow were found. The parasitological protection was associated with Th1-type cell response, since high IFN-γ, IL-12, and GM-CSF levels, in addition to low IL-4 and IL-10 production, were found. Liposome induced a better parasitological and immunological protection than did saponin. Experiments using PBMCs showed rLiHyE-stimulated lymphoproliferation in treated patients' and healthy subjects' cells, as well as high IFN-γ levels in the cell supernatant. In conclusion, rLiHyE could be considered for future studies as a vaccine candidate against VL.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Antígenos de Protozoos/administración & dosificación , Leishmania infantum/inmunología , Leishmaniasis Visceral/prevención & control , Animales , Antígenos de Protozoos/inmunología , Femenino , Humanos , Inmunogenicidad Vacunal , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/parasitología , Leucocitos Mononucleares/inmunología , Ratones , Ratones Endogámicos BALB C , Carga de Parásitos , Células TH1/inmunología , VacunaciónRESUMEN
Amphotericin B (Amp) has been well-successfully used to treat against Leishmania infection, although high toxicity has been found in patients. In the present study, Amp was administered in Leishmania infantum-infected BALB/c mice by three distinct delivery systems aiming to compare their efficacy against challenge infection, as well as their side effects in a murine visceral leishmaniasis (VL) model. This product was administered in a Poloxamer P407 (Pluronic® F127)-based polymeric micelle system (Amp/M), in the Ambisome® formulation (Lip-Amp) or in a free format (free Amp). Glucantime® (Gluc) was used as a comparative drug. Aiming to evaluate different endpoints of the treatments, the efficacy of the compounds was investigated one and 15-days after the therapeutic regimens, determining the parasite load by a limiting dilution assay and a quantitative PCR (qPCR) technique, as well as evaluating the immune response generated in the infected and treated animals. In the results, Amp/M or Lip-Amp-treated mice presented the best outcomes, since significant parasite load reductions were found in the evaluated organs, as well as a parasite-specific Th1 immune response was observed in the animals. In addition, no hepatic or renal damage was found in these mice. On the other hand, free Amp or Gluc induced toxicity in the animals, which was associated with a low Th1 immune response. Comparatively, Amp/M was the most effective drug in our experimental model, and results showed that the Amp-carrying system could be considered as a future alternative in studies against VL.
Asunto(s)
Anfotericina B/administración & dosificación , Antiprotozoarios/administración & dosificación , Sistemas de Liberación de Medicamentos/normas , Leishmaniasis Visceral/tratamiento farmacológico , Anfotericina B/toxicidad , Animales , Antiprotozoarios/toxicidad , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Riñón/efectos de los fármacos , Leishmania infantum/efectos de los fármacos , Hígado/efectos de los fármacos , Meglumina/administración & dosificación , Antimoniato de Meglumina , Ratones , Ratones Endogámicos BALB C , Micelas , Nitritos/metabolismo , Compuestos Organometálicos/administración & dosificación , Organismos Libres de Patógenos Específicos , Bazo/citología , Bazo/inmunologíaRESUMEN
Tegumentary leishmaniasis (TL) constitutes a major public health problem with significant morbidity worldwide. Synthetic peptide-based vaccines are attractive candidates to protect against leishmaniasis, since T cell-specific epitopes can be delivery to antigen-presenting cells, leading to the generation of a Th1 cell-mediated immunity. In this context, the present study aims to evaluate the immunogenicity and protective efficacy of a vaccine composed of major histocompatibility complex class I and II-restricted epitopes derived from four Leishmania infantum proteins to protect mice against Leishmania amazonensis infection. This recombinant fusion protein was administered in BALB/c mice alone or with saponin. As controls, animals received saline or saponin. In the results, the administration of the recombinant protein plus saponin induced a specific IFN-γ, IL-12 and GM-CSF production, as well as high IgG2a isotype antibody levels, which protected mice against a challenge using L. amazonensis promastigotes. Lower parasite burden was found in the infected footpads, liver, spleen and draining lymph node of vaccinated mice, when compared to those from the control groups. In addition, protection was associated with a lower IL-4 and IL-10 response, which was accompanied by the antileishmanial nitrite production by spleen cells of the animals. Interestingly, the recombinant protein administered alone induced a partial protection against challenge. In conclusion, this study shows a new vaccine candidate based on T cell-specific epitopes that was able to induce protection against L. amazonensis infection.
Asunto(s)
Leishmania infantum/inmunología , Vacunas contra la Leishmaniasis/inmunología , Leishmaniasis/inmunología , Proteínas Recombinantes de Fusión/inmunología , Vacunas de Subunidad/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Proteínas Bacterianas/genética , Citocinas/metabolismo , Epítopos de Linfocito T/genética , Femenino , Antígenos HLA/genética , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase II/genética , Humanos , Leishmaniasis/prevención & control , Ratones , Ratones Endogámicos BALB C , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Células TH1 , VacunaciónRESUMEN
In the present study, a conserved Leishmania hypothetical protein, namely LiHypA, was evaluated for the serodiagnosis of visceral and tegumentary leishmaniasis in dogs and humans. This protein showed a high amino acid sequence homology between viscerotropic and cutaneotropic Leishmania species. An enzyme-linked immunosorbent assay (ELISA) was developed using the recombinant antigen (rLiHypA), in addition to the A2 protein and two parasite antigenic preparations, which were used as controls. Regarding human diagnosis, results showed that rLiHypA was more sensitive and specific than ELISA-L. braziliensis SLA in detecting both cutaneous or mucosal leishmaniasis patients, but not those from Chagas disease patients or healthy subjects. Regarding canine diagnosis, this recombinant antigen showed higher sensitivity and specificity values, as well as a perfect accuracy to identify asymptomatic and symptomatic visceral leishmaniasis (VL) in dogs, but not those from vaccinated animals or those developing babesiosis, ehrlichiosis, or Chagas disease. However, using the rA2 protein or L. braziliensis SLA as controls, significant cross-reactivity was found when these samples were used, hampering their sensitivity and specificity values for the diagnosis. In this context, LiHypA could be considered a candidate to be evaluated for the serodiagnosis of visceral and tegumentary leishmaniasis in dogs and humans.
Asunto(s)
Antígenos de Protozoos/metabolismo , Enfermedad de Chagas/diagnóstico , Ensayo de Inmunoadsorción Enzimática/métodos , Leishmania/inmunología , Leishmaniasis Cutánea/diagnóstico , Leishmaniasis Visceral/diagnóstico , Proteínas Recombinantes/metabolismo , Animales , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Enfermedad de Chagas/inmunología , Secuencia Conservada/genética , Reacciones Cruzadas , Perros , Humanos , Leishmaniasis Cutánea/inmunología , Leishmaniasis Visceral/inmunología , Valor Predictivo de las Pruebas , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
A clioquinol (ICHQ)-containing Pluronic® F127 polymeric micelle system (ICHQ/Mic) was recently shown to be effective against Leishmania amazonensis infection in a murine model. In the present study, ICHQ/Mic was tested against L. infantum infection. BALB/c mice (n = 12 per group) were infected with L. infantum stationary promastigotes through subcutaneous injection and, 45 days after challenge, received saline or were treated via the subcutaneous route with empty micelles, ICHQ or ICHQ/Mic. In addition, animals were treated with miltefosine by the oral route, as a drug control. Half of the animals were euthanized 1 and 15 days after treatment, aiming to evaluate two endpoints after therapy, when parasitological and immunological parameters were investigated. Results showed that the treatment using miltefosine, ICHQ or ICHQ/Mic induced significantly higher anti-parasite IFN-γ, IL-12, GM-CSF, nitrite and IgG2a isotype antibody levels, which were associated with low IL-4 and IL-10 production. In addition, a higher frequency of IFN-γ and TNF-α-producing CD4+ and CD8+ T-cells was found in these animals. The parasite load was evaluated in distinct organs, and results showed that the treatment using miltefosine, ICHQ or ICHQ/Mic induced significant reductions in organic parasitism in the treated and infected mice. A comparison between the treatments suggested that ICHQ/Mic was the most effective in inducing a highly polarized Th1-type response, as well as reducing the parasite load in significant levels in the treated and infected animals. Data obtained 15 days after treatment suggested maintenance of the immunological and parasitological responses. In conclusion, ICHQ/Mic could be considered in future studies for the treatment of visceral leishmaniasis.
TITLE: Un système à micelles polymériques Pluronic® F127 contenant du clioquinol est efficace pour le traitement de la leishmaniose viscérale dans un modèle murin. ABSTRACT: Un système à micelles polymériques Pluronic® F127 (ICHQ/Mic) contenant du clioquinol (ICHQ) s'est récemment révélé efficace contre l'infection à Leishmania amazonensis dans un modèle murin. Dans la présente étude, l'ICHQ/Mic a été testé contre l'infection à L. infantum. Les souris BALB/c (n = 12 par groupe) ont été infectées par des promastigotes stationnaires de L. infantum par injection sous-cutanée et ont reçu 45 jours après l'épreuve une solution saline ou ont été traitées par voie sous-cutanée avec des micelles vides, ICHQ ou ICHQ/Mic. De plus, les animaux ont été traités avec de la miltefosine par voie orale, comme contrôle médicamenteux. La moitié des animaux ont été euthanasiés 1 et 15 jours après le traitement, dans le but de mesurer deux critères d'évaluation après la thérapie, lorsque les paramètres parasitologiques et immunologiques ont été étudiés. Les résultats ont montré que le traitement par miltefosine, ICHQ ou ICHQ/Mic induisait des niveaux d'anticorps anti-parasite IFN-γ, IL-12, GM-CSF, nitrite et IgG2a significativement plus élevés, associés à de faibles productions d'IL-4 et IL-10. De plus, une fréquence plus élevée de cellules T CD4+ et CD8+ produisant de l'IFN-γ and TNF-α a été trouvée chez ces animaux. La charge parasitaire a été évaluée dans des organes distincts et les résultats ont montré que le traitement utilisant la miltefosine, ICHQ ou ICHQ/Mic induisait des réductions significatives du parasitisme des organes chez les souris traitées et infectées. Une comparaison entre les traitements a suggéré qu'ICHQ/Mic était le plus efficace pour induire une réponse de type Th1 polarisée, ainsi que pour réduire la charge parasitaire à des niveaux significatifs chez les animaux traités et infectés. Les données obtenues 15 jours après le traitement suggèrent le maintien des réponses immunologiques et parasitologiques. En conclusion, ICHQ/Mic pourrait être envisagé dans de futures études pour le traitement contre la leishmaniose viscérale.
Asunto(s)
Clioquinol/uso terapéutico , Leishmaniasis Visceral/tratamiento farmacológico , Micelas , Poloxámero/química , Animales , Anticuerpos Antiprotozoarios/sangre , Clioquinol/química , Citocinas/inmunología , Modelos Animales de Enfermedad , Sistemas de Liberación de Medicamentos , Femenino , Leishmania infantum , Ratones , Ratones Endogámicos BALB C , Carga de Parásitos , Poloxámero/uso terapéutico , Células TH1/inmunologíaRESUMEN
In the present study, two Leishmania braziliensis proteins, one hypothetical and the eukaryotic initiation factor 5a (EiF5a), were cloned and used as a polyproteins vaccine for the heterologous protection of BALB/c mice against infantum infection. Animals were immunized with the antigens separately or in association, and in both cases saponin was used as an adjuvant. In the results, spleen cells from mice inoculated with the individual or polyproteins vaccine and lately challenged produced significantly higher levels of protein- and parasite-specific IFN-γ, IL-12, and GM-CSF, when both a capture ELISA and flow cytometry assays were performed. Evaluating the parasite load by a limiting dilution as well as by RT-PCR, these animals presented significant reductions in the parasite number in all evaluated organs, when compared to the control (saline and saponin) groups. The best protection was reached when the polyproteins vaccine was employed. Protection was associated with the IFN-γ production against parasite extracts, which was mediated by both CD4(+) and CD8(+) T cells and correlated with the antileishmanial nitrite production. In this context, this vaccine combining two L. braziliensis proteins was able to induce a heterologous protection against VL, and could be considered in future studies to be tested against other Leishmania species or in other mammalian hosts.