RESUMEN
The treatment of dogs naturally infected with Leishmania infantum using meglumine antimoniate (MA) encapsulated in conventional liposomes (LC) in association with allopurinol has been previously reported to promote a marked reduction in the parasite burden in the main infection sites. Here, a new assay in naturally infected dogs was performed using a novel liposome formulation of MA consisting of a mixture of conventional and long-circulating (PEGylated) liposomes (LCP), with expected broader distribution among affected tissues of the mononuclear phagocyte system. Experimental groups of naturally infected dogs were as follows: LCP plus Allop, receiving LCP intravenously as 2 cycles of 6 doses (6.5 mg Sb/kg of body weight/dose) at 4-day intervals plus allopurinol at 30 mg/kg/12 h per os (p.o.) during 130 days (LCP+Allop); LC plus Allop, receiving LC intravenously as 2 cycles of 6 doses (6.5 mg Sb/kg/dose) plus allopurinol during 130 days (LC+Allop); Allop, treated with allopurinol only; and a nontreated control. Parasite loads were evaluated by quantitative PCR in liver, spleen, and bone marrow tissue and by immunohistochemistry in the ear skin, before treatment, just after treatment, and 4 months later. The LCP+Allop and LC+Allop groups, but not the Allop group, showed significant suppression of the parasites in the liver, spleen, and bone marrow 4 months after treatment compared to the pretreatment period or the control group. Only LCP+Allop group showed significantly lower parasite burden in the skin in comparison to the control group. On the basis of clinical staging and parasitological evaluations, the LCP formulation exhibited a more favorable therapeutic profile than the LC one, being therefore promising for the treatment of canine visceral leishmaniasis.
Asunto(s)
Antiprotozoarios , Enfermedades de los Perros , Leishmania infantum , Leishmaniasis Visceral , Compuestos Organometálicos , Alopurinol/uso terapéutico , Animales , Antiprotozoarios/uso terapéutico , Enfermedades de los Perros/tratamiento farmacológico , Perros , Leishmaniasis Visceral/tratamiento farmacológico , Leishmaniasis Visceral/veterinaria , Liposomas/uso terapéutico , Meglumina/uso terapéutico , Antimoniato de Meglumina/uso terapéutico , Compuestos Organometálicos/uso terapéutico , Polietilenglicoles/uso terapéuticoRESUMEN
The protozoan Trypanosoma cruzi has the ability to spontaneously secrete extracellular vesicles (EVs). In this paper, T. cruzi EVs derived from epimastigote forms were evaluated during interaction with triatomine bugs Rhodnius prolixus and Triatoma infestans. T. cruzi EVs were purified and artificially offered to the insects prior to infection with epimastigote forms. No effect of EVs was detected in the parasite counts in the guts of both vectors after 49-50 days. On the other hand, pre-feeding with EVs delayed parasite migration to rectum only in the gut in R. prolixus after 21-22 days. Those data suggest a possible role of T. cruzi EVs during the earlier events of infection in the invertebrate host.
Asunto(s)
Vesículas Extracelulares , Insectos Vectores/parasitología , Intestinos/parasitología , Rhodnius/parasitología , Triatoma/parasitología , Trypanosoma cruzi/fisiología , Animales , Interacciones Huésped-Parásitos/fisiología , Trypanosoma cruzi/citologíaRESUMEN
The need for daily parenteral administration is an important limitation in the clinical use of pentavalent antimonial drugs against leishmaniasis. In this study, amphiphilic antimony(V) complexes were prepared from alkylmethylglucamides (L8 and L10, with carbon chain lengths of 8 and 10, respectively), and their potential for the oral treatment of visceral leishmaniasis (VL) was evaluated. Complexes of Sb and ligand at 1:3 (SbL8 and SbL10) were obtained from the reaction of antimony(V) with L8 and L10, as evidenced by elemental and electrospray ionization-tandem mass spectrometry (ESI-MS) analyses. Fluorescence probing of hydrophobic environment and negative-staining transmission electron microscopy showed that SbL8 forms kinetically stabilized nanoassemblies in water. Pharmacokinetic studies with mice in which the compound was administered by the oral route at 200 mg of Sb/kg of body weight indicated that the SbL8 complex promoted greater and more sustained Sb levels in serum and liver than the levels obtained for the conventional antimonial drug meglumine antimoniate (Glucantime [Glu]). The efficacy of SbL8 and SbL10 administered by the oral route was evaluated in BALB/c mice infected with Leishmania infantum after a daily dose of 200 mg of Sb/kg for 20 days. Both complexes promoted significant reduction in the liver and spleen parasite burdens in relation to those in the saline-treated control group. The extent of parasite suppression (>99.96%) was similar to that achieved after Glu given intraperitoneally at 80 mg of Sb/kg/day. As expected, there was no significant reduction in the parasitic load in the group treated orally with Glu at 200 mg of Sb/(kg day). In conclusion, amphiphilic antimony(V) complexes emerge as an innovative and promising strategy for the oral treatment of VL.
RESUMEN
An innovative liposomal formulation of meglumine antimoniate (LMA) was recently reported to promote both long-term parasite suppression and reduction of infectivity to sand flies in dogs with visceral leishmaniasis. However, 5 months after treatment, parasites were still found in the bone marrow of all treated dogs. In order to improve treatment with LMA, the present study aimed to evaluate its efficacy in combination with allopurinol. Mongrel dogs naturally infected with Leishmania infantum were treated with six doses of LMA (6.5 mg Sb/kg of body weight/dose) given at 4-day intervals, plus allopurinol (20 mg/kg/24 h per os) for 140 days. Comparison was made with groups treated with LMA, allopurinol, empty liposomes plus allopurinol, empty liposomes, and saline. Dogs remained without treatment from day 140 to 200 after the start of treatment. The drug combination promoted both clinical improvement of dogs and significant reduction in the parasitic load in bone marrow and spleen on days 140 and 200 compared to these parameters in the pretreatment period. This is in contrast with the other protocols, which did not result in significant reduction of the bone marrow parasite load on day 200. Strikingly, the combined treatment, in contrast to the other regimens, induced negative quantitative PCR (qPCR) results in the liver of 100% of the dogs. Both xenodiagnosis and skin parasite determination by qPCR indicated that the drug combination was effective in blocking the transmission of skin parasites to sand flies. Based on all of the parasitological tests performed on day 200, 50% of the animals that received the combined treatment were considered cured.
Asunto(s)
Alopurinol/química , Alopurinol/uso terapéutico , Antiprotozoarios/uso terapéutico , Leishmaniasis Visceral/tratamiento farmacológico , Liposomas/química , Meglumina/química , Meglumina/uso terapéutico , Compuestos Organometálicos/química , Compuestos Organometálicos/uso terapéutico , Animales , Antiprotozoarios/química , Perros , Femenino , Masculino , Antimoniato de MegluminaRESUMEN
Experimental vaccines to protect against visceral leishmaniasis (VL) have been developed by using BALB/c mice infected with a large (107 to 108) inoculum of parasites. Remarkably, prior literature has reported that the poor protection observed is mainly due to the high susceptibility of this strain. To determine factors inherent to mice that might abrogate vaccine-induced efficacy, the present research sought to investigate the impact of the administration of different infective inoculums of Leishmania chagasi (syn. L. infantum) in BALB/c mice, evaluating subcutaneous and intravenous routes of administration as well as parasitological and immunological parameters over different periods of time. This study shows that the injection of a highly infective inoculum in mice, through both subcutaneous and intravenous routes, results in a sustained infection. The mice developed a high parasite load in the liver; however, these values diminished over time. This result did not corroborate with the parasite load in the bone marrow and brain and proved to be expressively different in the spleen and draining lymph nodes, where the values increased over time. Mice infected with a low dose of parasites (10³) showed a certain resistance against infection, based mainly on the IFN-γ and oxide nitric production. Considering all the elements, it could be concluded that the models employing high doses (107) of L. chagasi in BALB/c mice can bring about an imbalance in the animals' immune response, thus allowing for the development of the disease at the expense of efficacy within the vaccine candidates.
Asunto(s)
Leishmania infantum/fisiología , Leishmania infantum/patogenicidad , Leishmaniasis Visceral/inmunología , Animales , Anticuerpos Antiprotozoarios/sangre , Citocinas/biosíntesis , Femenino , Inyecciones Intravenosas , Inyecciones Subcutáneas , Leishmaniasis Visceral/parasitología , Ratones , Ratones Endogámicos BALB C , Óxido Nítrico/biosíntesis , Carga de Parásitos , Factores de TiempoRESUMEN
Two novel trivalent antimony(III) and bismuth(III) complexes with the nitrogen-donor heterocyclic ligand dipyrido[3,2-a:2',3'-c]phenazine (dppz) were synthesized and characterized as [Sb(dppz)Cl3]âH2OâCH3OH and [Bi(dppz)Cl3]. The crystal structure of Sb(III) complex was determined by X-ray crystallography. These complexes were evaluated for their activity against the promastigote form of Sb(III)-sensitive and -resistant Leishmania infantum chagasi and Leishmania amazonensis strains. Both complexes were more effective than dppz alone in inhibiting the growth of Leishmania promastigotes and were at least 77 and 2,400 times more active than potassium antimonyl tartrate in Sb(III)-sensitive and -resistant Leishmania, respectively. The cytotoxicity of dppz and its complexes against mouse peritoneal macrophages occurred at dppz concentrations at least 6-fold greater than those found to be active against Leishmania promastigotes.To investigate the role of the metal in the improved antileishmanial activity of dppz, the activity of the Sb(III) complex was compared between the Sb-resistant mutants and their respective parental sensitive strains. The lack of cross-resistance to the Sb(III)-dppz complex together with the much lower activity of antimonyl tartrate, SbCl3 and BiCl3 strongly support the model that the metal is not active by itself but improves the activity of dppz through complexation.
Asunto(s)
Antimonio/química , Bismuto/química , Complejos de Coordinación/farmacología , Leishmania/efectos de los fármacos , Fenazinas/química , Tripanocidas/farmacología , Animales , Antimonio/farmacología , Bismuto/farmacología , Células Cultivadas , Complejos de Coordinación/química , Resistencia a Medicamentos , Concentración 50 Inhibidora , Macrófagos Peritoneales/efectos de los fármacos , Ratones , Estructura Molecular , Fenazinas/farmacología , Espectroscopía Infrarroja por Transformada de Fourier , Tripanocidas/químicaRESUMEN
Hyperactivation of tubular cells contributes for the progression of kidney lesions. The exacerbated expression of immunological proteins and ribosomal DNA (rDNA) transcriptional activity are observed in tubular cells. This intensified expression results in more prominent hypertrophic changes and is often accompanied by increased expression of factors involved in different phases of ribosomal biosynthesis, such as the nucleolar organizer regions (NOR). The aim of this study was to evaluate whether there is an association between NOR proteins, renal impairment, and clinical status in Leishmania-infected dogs (CanL). Forty-five dogs with CanL and six uninfected controls were assessed in this study. PCR was performed to detect parasites' nucleic acids in kidney. Histopathological analyses were performed in kidney fragments, and NOR was detected by Ag stain (AgNOR). Leishmania-infected dogs showed more intense inflammation and collagen deposition compared with uninfected controls. Biochemical alterations were observed only in Leishmania-infected dogs. AgNORs per cell were significantly higher in clinically affected dogs and higher histopathological lesion score was observed in Leishmania-infected dogs. Positive correlations between number of NORs per cell in medullary region and histopathological lesion score were observed. Furthermore, AgNOR expression, intensity of renal lesions, and clinical sigs was associated in Leishmania-infected dogs. We propose that the detection of AgNOR proteins could be used to better estimate the kidney tubular damage at the time of examination in Leishmania-infected dogs as a marker to estimate renal impairment in dogs with CanL.
Asunto(s)
Enfermedades de los Perros , Leishmania infantum , Insuficiencia Renal , Animales , Enfermedades de los Perros/diagnóstico , Perros , Riñón , Región Organizadora del Nucléolo , Insuficiencia Renal/veterinariaRESUMEN
Identification of novel antigens is essential for developing new diagnostic tests and vaccines. We used DIGE to compare protein expression in amastigote and promastigote forms of Leishmania chagasi. Nine hundred amastigote and promastigote spots were visualized. Five amastigote-specific, 25 promastigote-specific, and 10 proteins shared by the two parasite stages were identified. Furthermore, 41 proteins were identified in the Western blot employing 2-DE and sera from infected dogs. From these proteins, 3 and 38 were reactive with IgM and total IgG, respectively. The proteins recognized by total IgG presented different patterns in terms of their recognition by IgG1 and/or IgG2 isotypes. All the proteins selected by Western blot were mapped for B-cell epitopes. One hundred and eighty peptides were submitted to SPOT synthesis and immunoassay. A total of 25 peptides were shown of interest for serodiagnosis to visceral leishmaniasis. In addition, all proteins identified in this study were mapped for T cell epitopes by using the NetCTL software, and candidates for vaccine development were selected. Therefore, a large-scale screening of L. chagasi proteome was performed to identify new B and T cell epitopes with potential use for developing diagnostic tests and vaccines.
Asunto(s)
Antígenos de Protozoos/inmunología , Electroforesis en Gel Bidimensional/métodos , Epítopos de Linfocito B/genética , Leishmania/inmunología , Leishmaniasis Visceral/diagnóstico , Proteómica/métodos , Pruebas Serológicas/métodos , Animales , Western Blotting , Biología Computacional , Perros , Inmunoensayo , Especificidad de la Especie , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
This work reports results of re-infection of BALB/c and C57BL/6 mice with different recombinant strains of Toxoplasma gondii. Mice were prime-infected with the non-virulent D8 strain and challenged with virulent strains. PCR-RFLP of cS10-A6 genetic marker of T. gondii demonstrated that BALB/c mice were re-infected with the EGS strain, while C57BL/6 mice were re-infected with the EGS and CH3 strains. Levels of IFN-γ and IL-10 after D8 prime-infection were lower in C57BL/6 than in BALB/c mice. Brain inflammation after D8 prime-infection was more intense in C57BL/6 than in BALB/c mice. It was shown that re-infection depends on mice lineage and genotype of the strain used in the challenge.
Asunto(s)
Toxoplasma/inmunología , Toxoplasmosis Animal/inmunología , Animales , Bioensayo , Encéfalo/parasitología , Encéfalo/patología , Células Cultivadas , Pollos , Citocinas/análisis , Perros , Femenino , Genotipo , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Recurrencia , Bazo/citología , Bazo/inmunología , Toxoplasma/clasificación , Toxoplasma/genéticaRESUMEN
The pathogenesis of Canine leishmaniosis (CanL) is associated with altered cytokine expression and parasitic tissue shows a lot of inflammation. The aim of this study was to assess the renal inflammation and cytokine expression in eight symptomatic and eight asymptomatic Leishmania- infected dogs, and seven uninfected control dogs. Kidney fragments were stained with hematoxylin and eosin for morphometric evaluation. mRNA expression levels of interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin (IL)-2, IL-4, IL-10, and IL-12 were assessed in the kidney fragments using quantitative real time-polymerase chain reaction. Inflammation, quantified by the average area of the infiltrated immune cells, was greater in symptomatic dogs than in those asymptomatic, whereas asymptomatic dogs exhibited higher inflammation than the control dogs (p > 0.05, Tukey's test). Expression levels of IFN-γ, TNF-α, IL-4, IL-10, and IL-12 were upregulated in symptomatic dogs and downregulated in asymptomatic dogs compared with those of the uninfected group. Furthermore, IL-4 showed higher expression in symptomatic dogs than in asymptomatic ones (p < 0.05, Mann-Whitney test), which was directly associated with clinical manifestations (p < 0.05, Chi-square test). However, IL-12 was predominantly expressed in symptomatic dogs, shifting the balance from IL-12/IL-4 to IL-12, which elicits a change in the inflammatory response. Leishmania was not found in the renal tissues in any one of the studied groups. Our data suggests that the balance between IL-12 and IL-4 plays an important role in the regulation of inflammation in renal tissue and clinical presentations in CanL.
Asunto(s)
Enfermedades de los Perros/inmunología , Inflamación/veterinaria , Interleucina-12/genética , Interleucina-4/genética , Riñón/inmunología , Leishmaniasis/inmunología , Leishmaniasis/veterinaria , Animales , Enfermedades de los Perros/parasitología , Perros , Femenino , Regulación de la Expresión Génica/inmunología , Inflamación/parasitología , Interleucina-12/inmunología , Interleucina-4/inmunología , Leishmania infantum/inmunología , MasculinoRESUMEN
Visceral leishmaniasis (VL) is a systemic parasitic disease that leads to high rates of morbidity and mortality in humans worldwide. There is a great need to develop new drugs and novel strategies to make chemotherapy for this disease more efficacious and well tolerated. Recent reports on the immunomodulatory effects and the low toxicity of the spherical carbon nanostructure fullerol led us to investigate in vitro and in vivo antileishmanial activity in free and encapsulated forms in liposomes. When assayed against intramacrophagic Leishmania amastigotes, fullerol showed a dose-dependent reduction of the infection index with IC50 of 0.042â¯mg/mL. When given daily by i.p. route for 20 days (0.05â¯mg/kg/d) in a murine model of acute VL, fullerol promoted significant reduction in the liver parasite load. To improve the delivery of fullerol to the infection sites, liposomal formulations were prepared by the dehydration-rehydration method. When evaluated in the acute VL model, liposomal fullerol (Lip-Ful) formulations given i.p. at 0.05 and 0.2â¯mg/kg with 4-days intervals were more effective than the free form, with significant parasite reductions in both liver and spleen. Lip-Ful at 0.2â¯mg/kg promoted complete parasite elimination in the liver. The antileishmanial activity of Lip-Ful was further confirmed in a chronic model of VL. Lip-Ful was also found to induce secretion of pro-inflammatory TNF-α, IFN-γ and IL-1ß cytokines. In conclusion, this work reports for the first time the antileishmanial activity of fullerol and introduces an innovative approach for treatment of VL based on the association of this nanostructure with liposomes.
Asunto(s)
Fulerenos/farmacología , Leishmania infantum/efectos de los fármacos , Leishmania mexicana/efectos de los fármacos , Leishmaniasis Visceral/tratamiento farmacológico , Lípidos/química , Hígado/parasitología , Macrófagos Peritoneales/parasitología , Tripanocidas/farmacología , Animales , Citocinas/sangre , Modelos Animales de Enfermedad , Composición de Medicamentos , Femenino , Fulerenos/química , Mediadores de Inflamación/sangre , Leishmania infantum/crecimiento & desarrollo , Leishmania mexicana/crecimiento & desarrollo , Leishmaniasis Visceral/sangre , Leishmaniasis Visceral/parasitología , Liposomas , Hígado/metabolismo , Mesocricetus , Ratones Endogámicos BALB C , Nanopartículas , Carga de Parásitos , Tripanocidas/químicaRESUMEN
Nramp1 (Slc11a1) is linked to resistance to Leishmania in mice, but its role in canine leishmaniasis is not clear. In this study we sequenced the Nramp1 cDNA from dogs whose macrophages allowed or restricted intracellular growth of Leishmania chagasi. Peripheral blood monocyte-derived macrophages were isolated from 29 dogs, cultured and inoculated with L. chagasi. This approach resulted in the identification of dogs whose macrophages were resistant or susceptible to L. chagasi. Nramp1 cDNA sequences of these dogs were identical. mRNA levels of Nramp1, IFNgamma, IL-4 and the subunit p35 of IL-12 were assessed in the spleen of naturally infected symptomatic and asymptomatic dogs in comparison to uninfected controls. Although not statistically significant, asymptomatic dogs had a tendency for higher levels of Nramp1 mRNA (p = 0.11). Expression of Nramp1 was then compared between phenotypically resistant and susceptible dogs, without any significant difference between these groups.
Asunto(s)
Proteínas de Transporte de Catión/genética , ADN Complementario/genética , Enfermedades de los Perros/genética , Predisposición Genética a la Enfermedad , Leishmaniasis Visceral/veterinaria , Animales , Secuencia de Bases , Proteínas de Transporte de Catión/inmunología , Perros , Regulación de la Expresión Génica/inmunología , Leishmaniasis Visceral/genética , Leishmaniasis Visceral/inmunología , Macrófagos/parasitologíaRESUMEN
American visceral leishmaniasis is a zoonosis of the New World. Dogs are the main reservoir of the disease and there is much interest in the understanding of mechanisms implicated in protection against canine infection. Nevertheless, most studies in dogs have not been carried out in organs that are targets of infection. This work is first to report the profile of cytokines and parasite burdens, as determined by real-time PCR, in the lymph nodes of dogs naturally infected with Leishmania chagasi. With this purpose, 18 mongrel dogs were divided in three groups: control non-infected dogs (n=6) and naturally infected animals with L. chagasi, asymptomatic (n=6) and symptomatic (n=6). Parasite burden in lymph nodes was 73-fold greater in symptomatic than asymptomatic animals. Prescapular lymph nodes of asymptomatic dogs had the highest expression of IFN-gamma and TNF-alpha and low parasite burden, indicating that these cytokines play a role in protection against infection. Highest expression of IL-10 and TGF-beta and high parasite burden were observed in symptomatic dogs, suggesting a role for these cytokines in the progression of disease. Hence, the balance of expression of IFN-gamma and TNF-alpha (protective) and IL-10 and TGF-beta (disease progression) in lymph nodes determine parasite burden and clinical expression in naturally infected dogs.
Asunto(s)
Citocinas/biosíntesis , Enfermedades de los Perros/inmunología , Enfermedades de los Perros/parasitología , Leishmania/inmunología , Leishmaniasis Visceral/veterinaria , Ganglios Linfáticos/parasitología , Animales , Citocinas/genética , Citocinas/inmunología , ADN Protozoario/análisis , Perros , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inmunohistoquímica/veterinaria , Leishmania/genética , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/parasitología , Ganglios Linfáticos/inmunología , Masculino , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Estadísticas no Paramétricas , Zoonosis/parasitologíaRESUMEN
The toxicity and antileishmanial effectiveness of a novel liposome formulation of meglumine antimoniate in mongrel dogs with visceral leishmaniasis (VL) obtained from a region where VL is endemic in Brazil have been investigated. Groups of 12 animals received by the intravenous route four doses (with 4-day intervals) of either liposomal meglumine antimoniate (group I [GI], 6.5 mg Sb/kg of body weight/dose), empty liposomes (GII), or isotonic saline (GIII). Evaluation of markers of hematopoietic, hepatic, and renal functions before and just after treatment showed no significant change. On the other hand, transitory adverse reactions, including prostration, defecation, tachypnea, and sialorrhea, were observed during the first 15 min after injections in GI and GII. Parasitological evaluation of sternal bone marrow 4 days after the last dose showed a significant reduction of parasite burden in GI, compared to the other groups. Immunocytochemical evaluations of the skin, bone marrow, cervical lymph nodes, livers, and spleens of dogs for parasites, 150 days after treatment, indicated significant parasite suppression (higher than 95.7%) in the lymph nodes, livers, and spleens of GI, compared to control groups. Feeding of Lutzomyia longipalpis phlebotomines on dogs from GI, 150 days after treatment, resulted in a significant reduction of sand fly infection efficiency, compared to feeding on animals from GII and GIII. This is the first report of both long-term parasite suppression and reduction of infectivity to sand flies in naturally infected dogs following treatment with a liposome-encapsulated drug. Importantly, this was achieved using a 20-fold-lower cumulative dose of Sb than is used for conventional antimonial treatment.
Asunto(s)
Antiprotozoarios/administración & dosificación , Enfermedades de los Perros/tratamiento farmacológico , Leishmania infantum , Leishmaniasis Visceral/veterinaria , Meglumina/administración & dosificación , Compuestos Organometálicos/administración & dosificación , Phlebotomus/parasitología , Animales , Antiprotozoarios/toxicidad , Enfermedades de los Perros/parasitología , Enfermedades de los Perros/transmisión , Perros , Femenino , Insectos Vectores/parasitología , Leishmania infantum/efectos de los fármacos , Leishmania infantum/aislamiento & purificación , Leishmania infantum/patogenicidad , Leishmaniasis Visceral/tratamiento farmacológico , Leishmaniasis Visceral/parasitología , Leishmaniasis Visceral/transmisión , Liposomas , Masculino , Meglumina/toxicidad , Antimoniato de Meglumina , Compuestos Organometálicos/toxicidadRESUMEN
BACKGROUND: There are a few works considering the characterization of canine monocyte-derived macrophages as well as a standardized procedure for isolation, culture, and infection of these cells with Leishmania. We have performed several modifications in order to improve the canine monocyte-derived macrophage cultures. In addition, we have done a comparative study between monocytes and monocyte-derived macrophages from dogs naturally and experimentally infected with L. chagasi. RESULTS: In the presence of exogenous serum, opsonized Leishmania promastigotes binds better to monocytes/macrophages than without serum. Otherwise, this binding occurs due to the strict correlation between the opsonized biologic particles with the third receptor of the complement (CR3-CD11b/CD18). In fact, our assays with CD11b confirmed the importance of this receptor for canine cells and the L. chagasi experimental system. Moreover, monocytes obtained from naturally infected dogs have shown a higher number of monocytes bounded to promastigotes. The experimental results regarding survival have shown that promastigote forms of opsonized L. chagasi were more infective, because we found higher numbers of promastigotes bound to the different cells. As a consequence, after forty-eight hours of binding, higher numbers of amastigotes appeared inside monocyte-macrophages. CONCLUSION: These studies have given support to continue comparative studies involving canine monocytes, monocyte-derived macrophages and peritoneal macrophages. Since we have standardized the canine cell culture, we are looking forward to determining the phenotypic properties of these cells before and after L. chagasi infection using flow cytometry.
Asunto(s)
Enfermedades de los Perros/parasitología , Leishmania infantum/metabolismo , Leishmaniasis Visceral/veterinaria , Monocitos/parasitología , Animales , Enfermedades de los Perros/metabolismo , Perros , Femenino , Citometría de Flujo/veterinaria , Leishmaniasis Visceral/parasitología , Macrófagos/metabolismo , Macrófagos/parasitología , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/parasitología , Masculino , Monocitos/metabolismo , Análisis de SupervivenciaRESUMEN
The growing resistance of leishmaniasis to first-line drugs like antimonials in some regions limits the control of this parasitic disease. The precise mechanisms involved in Leishmania antimony resistance are still subject to debate. The reduction of intracellular SbIII accumulation is a common change observed in both laboratory-selected and field isolated resistant Leishmania strains, but the exact transport pathways involved in antimony resistance have not yet been elucidated. In order to functionally characterize the antimony transport routes responsible for resistance, we performed systematic transport studies of SbIII in wild-type and resistant strains of L. (Viannia) guyanensis and L. (V.) braziliensis. Those include influx and efflux assays and the influence of ABC transporters and metabolism inhibitors: prochlorperazine, probenecid, verapamil, BSO, and sodium azide. The mRNA levels of genes associated with antimony resistance (MRPA, GSH1, ODC, AQP1, ABCI4, and ARM58) were also investigated in addition to intracellular thiol levels. A strong reduction of Sb influx was observed in L. guyanensis resistant mutant (LgSbR), but not in L. braziliensis (LbSbR). Both mutants showed increased energy-dependent efflux of SbIII, when compared to their respective parental strains. In LgSbR, BSO and prochlorperazine inhibited antimony efflux and resistance was associated with increased MRPA and GSH1 mRNA levels, while in LbSbR antimony efflux was inhibited by probenicid and prochlorperazine in absence of resistance-associated gene modulation. Intracellular thiol levels were increased in both Sb-resistant mutants. An energy-dependent SbIII efflux pathway sensitive to prochlorperazine was clearly evidenced in both Sb-resistant mutants. In conclusion, the present study allowed the biophysical and pharmacological characterization of energy-dependent Sb efflux pathway apparently independent of MRPA, ABCI4, and ARM58 upregulation, in Leishmania (Vianna) mutant selected in vitro for resistance to SbIII. Prochlorperazine has also been identified as an effective chemosensitizer in both Sb resistant mutants, which acts through inhibition of the active efflux of Sb.
RESUMEN
American tegumentary leishmaniasis (ATL) is a neglected disease widely distributed in Latin America. In Brazil, it is caused by different Leishmania species belonging to the Subgenera Viannia and Leishmania. ATL diagnosis is routinely based on clinical, epidemiological, parasitological and immunological (delayed-type hypersensitivity skin test-DTH) evidences. The main objective of this work was to determine the efficacy of a previous immunohistochemical (IHC) method developed by our group. Seventy eight skin biopsies from patients with different ATL clinical forms and origins were evaluated. The method was previously standardized in ATL patients from the municipality of Caratinga, Minas Gerais, Brazil, all infected with Leishmania (V.) braziliensis. Here, it is evaluated in patients from the North, Southeast and Midwest regions of Brazil. Clinical, parasitological (biopsy PCR) and immunological (Montenegro skin test-MST) diagnosis were performed prior to IHC procedure. The IHC procedure detected 70.5% of the cases having a high agreement with MST diagnosis (kappa=0.84). A distinguished contribution of this work is that IHC succeed in diagnosing some negative DTH patients. Those were infected with Leishmania (L.) amazonensis, commonly causing the anergic form of the disease. In conclusion, IHC succeed in detecting ATL caused by different Leishmania species from various geographic regions and clinical status. Although it was not able to detect ATL in all patients, it was better than MST providing an additional tool for the diagnosis of ATL patients. There was no significant correlation between clinical forms and histological features including the presence of necrosis.
Asunto(s)
Inmunohistoquímica , Leishmania/aislamiento & purificación , Leishmaniasis Cutánea/diagnóstico , Leishmaniasis Cutánea/parasitología , Piel/parasitología , Adolescente , Adulto , Anciano , Biopsia , Brasil/epidemiología , Femenino , Humanos , Leishmania/genética , Leishmania/inmunología , Leishmania braziliensis/inmunología , Leishmania braziliensis/aislamiento & purificación , Leishmaniasis Cutánea/epidemiología , Hígado/parasitología , Hígado/patología , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Piel/patología , Piel/ultraestructura , Estados Unidos , Adulto JovenRESUMEN
A novel liposomal formulation of meglumine antimoniate (MA), consisting of vesicles of reduced size, has been evaluated in dogs with visceral leishmaniasis to determine its pharmacokinetics as well as the impact of vesicle size on the targeting of antimony to the bone marrow. Encapsulation of MA in liposomes was achieved through freeze-drying of empty liposomes in the presence of sucrose and rehydration with a solution of MA. The resulting formulation, with a mean vesicle diameter of about 400 nm, was given to mongrel dogs with visceral leishmaniasis as an i.v. bolus injection at 4.2 mgSb/kg of body weight. The pharmacokinetics of antimony were assessed in the blood and in organs of the mononuclear phagocyte system and compared to those achieved with the free drug and the drug encapsulated in large sized liposomes (mean diameter of 1200 nm). The targeting of antimony to the bone marrow was improved (approximately three-fold) with the novel liposomal formulation, when compared to the formulation of MA in large sized liposomes. This study provides the first direct experimental evidence that passive targeting of liposomes to the bone marrow of dogs is improved by the reduction of vesicle size from the micron to the nanometer scale.
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Antimonio/administración & dosificación , Médula Ósea , Sistemas de Liberación de Medicamentos , Animales , Antimonio/farmacocinética , Perros , Liposomas , Meglumina/administración & dosificación , Antimoniato de Meglumina , Compuestos Organometálicos/administración & dosificación , Tamaño de la Partícula , Distribución TisularRESUMEN
There is a great need for orally active drugs for the treatment of the neglected tropical disease leishmaniasis. Amphiphilic Sb(V) complexes, such as 1:3 Sb-N-octanoyl-N-methylglucamide complex (SbL8), are promising drug candidates. It has been previously reported that SbL8 forms kinetically stabilized nanoassemblies in water and that this simple dispersion exhibits antileishmanial activity when given by oral route to a murine model of visceral leishmaniasis. The main objective of the present work was to interfere in the structural organization of these nanoassemblies so as to investigate their influence on the oral bioavailability of Sb, and ultimately, optimize an oral formulation of SbL8 for the treatment of cutaneous leishmaniasis. The structural organization of SbL8 nanoassemblies was manipulated through addition of propylene glycol (PG) to the aqueous dispersion of SbL8. The presence of 50% (v/v) PG resulted in the loss of hydrophobic microenvironment, as evidenced by fluorescence probing. However, nanostructures were still present, as demonstrated by dynamic light scattering, small-angle X-ray scattering, and atomic force microscopy (AFM). A remarkable property of these nanoassemblies, as revealed by AFM analysis, is the flexibility of their supramolecular organization, which showed changes as a function of the solvent and substrate polarities. The formulation of SbL8 in 1:1 water:PG given orally to mice promoted significantly higher and more sustained serum levels of Sb, when compared to SbL8 in water. The new formulation, when given as repeated doses (200 mg Sb/kg/day) to BALB/c mice infected with Leishmania amazonensis, was significantly more effective in reducing the lesion parasite burden, compared to SbL8 in water, and even, the conventional drug Glucantime(®) given intraperitoneally at the same dose. In conclusion, this work introduces a new concept of polarity-sensitive nanocarrier that was successfully applied to optimize an oral formulation of Sb(V) for treating cutaneous leishmaniasis.
Asunto(s)
Antimonio/uso terapéutico , Antiprotozoarios/uso terapéutico , Portadores de Fármacos/química , Leishmaniasis Cutánea/tratamiento farmacológico , Nanopartículas/química , Administración Oral , Animales , Antimonio/sangre , Antimonio/farmacocinética , Antiprotozoarios/química , Antiprotozoarios/farmacocinética , Modelos Animales de Enfermedad , Femenino , Interacciones Hidrofóbicas e Hidrofílicas , Meglumina/uso terapéutico , Antimoniato de Meglumina , Ratones Endogámicos BALB C , Microscopía de Fuerza Atómica , Nanopartículas/ultraestructura , Compuestos Organometálicos/uso terapéutico , Dispersión del Ángulo Pequeño , Solventes , Resultado del Tratamiento , Difracción de Rayos XRESUMEN
The double stranded RNA (dsRNA) virus Leishmaniavirus (Totiviridae) was first described in Leishmania guyanensis and L. braziliensis (LRV1), and more recently from L. major and L. aethiopica (LRV2). Parasites bearing LRV1 elicit a higher pro-inflammatory profile, arising through activation of Toll like receptor 3(TLR3) interacting with the viral dsRNA. LRV1 is most common in Leishmania from the Amazon region; however data for other regions of Brazil are more limited. Here we applied PCR tests with validated 'universal' LRV1 primers to search for LRV1 in 40 strains of cultured L. braziliensis from several locales within Minas Gerais State, including patients presenting with atypical lesion pathology. All strains were negative however. These data are in agreement with results from other areas of Southeastern Brazil that LRV1 is relatively uncommon.