Nucleolar organizer region proteins enhancement in nucleoplasm's of renal tubular cells is an indication of kidney impairment in Leishmania-infected dogs.

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.

The balance between IL-12/IL4 in renal tissue switches the inflammatory response arm and shows relationship with the clinical signs in Leishmania-infected dogs.

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.

Antileishmanial activity of fullerol and its liposomal formulation in experimental models of visceral leishmaniasis.

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 0.042 mg/mL. When given daily by i.p. route for 20 days (0.05 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 0.2 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 0.2 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.

Therapeutic Efficacy of a Mixed Formulation of Conventional and PEGylated Liposomes Containing Meglumine Antimoniate, Combined with Allopurinol, in Dogs Naturally Infected with Leishmania infantum.

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.

Extracellular vesicles isolated from Trypanosoma cruzi affect early parasite migration in the gut of Rhodnius prolixus but not in Triatoma infestans.

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.

Biophysical and Pharmacological Characterization of Energy-Dependent Efflux of Sb in Laboratory-Selected Resistant Strains of Leishmania (Viannia) Subgenus.

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.

Effectiveness of an immunohistochemical protocol for Leishmania detection in different clinical forms of American tegumentary leishmaniasis.

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.

Lipophosphoglycans from Leishmania amazonensis Strains Display Immunomodulatory Properties via TLR4 and Do Not Affect Sand Fly Infection.

The immunomodulatory properties of lipophosphoglycans (LPG) from New World species of Leishmania have been assessed in Leishmania infantum and Leishmania braziliensis, the causative agents of visceral and cutaneous leishmaniasis, respectively. This glycoconjugate is highly polymorphic among species with variation in sugars that branch off the conserved Gal(ß1,4)Man(α1)-PO4 backbone of repeat units. Here, the immunomodulatory activity of LPGs from Leishmania amazonensis, the causative agent of diffuse cutaneous leishmaniasis, was evaluated in two strains from Brazil. One strain (PH8) was originally isolated from the sand fly and the other (Josefa) was isolated from a human case. The ability of purified LPGs from both strains was investigated during in vitro interaction with peritoneal murine macrophages and CHO cells and in vivo infection with Lutzomyia migonei. In peritoneal murine macrophages, the LPGs from both strains activated TLR4. Both LPGs equally activate MAPKs and the NF-κB inhibitor p-IκBα, but were not able to translocate NF-κB. In vivo experiments with sand flies showed that both stains were able to sustain infection in L. migonei. A preliminary biochemical analysis indicates intraspecies variation in the LPG sugar moieties. However, they did not result in different activation profiles of the innate immune system. Also those polymorphisms did not affect infectivity to the sand fly.

Low frequency of LRV1 in Leishmania braziliensis strains isolated from typical and atypical lesions in the State of Minas Gerais, Brazil.

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.

Polarity-sensitive nanocarrier for oral delivery of Sb(V) and treatment of cutaneous leishmaniasis.

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.

Genetic differences between two Leishmania major-like strains revealed by suppression subtractive hybridization.

Leishmania major, the causative agent of zoonotic leishmaniasis, is restricted to Old World countries. Molecular and biochemical techniques have been used to identify some L. major-like isolated in South America including Brazil. Here, two L. major-like strains, one virulent (BH49) and one non-virulent (BH121), were subjected to suppression subtractive hybridization (SSH) technique in order to identify differentially expressed genes. SSH technique identified nine cDNA fragments exhibiting high homology to previously sequenced L. major genes. Five cDNAs (four specific for BH49 and one for BH121) were confirmed by RT-PCR. Among those differentially expressed subtracted genes, some were involved in physiological processes including metabolism, translation and destination of proteins, production of energy, virulence factors and unknown functions. Western-blot analysis confirmed a higher expression level of ß-1,3-galactosyl residues in L. major-like lipophosphoglycan (LPG). This molecular analysis opens the possibility for identification of potential virulence factors not only in different strains, but also in others species of Leishmania.

Differentially expressed genes of virulent and nonvirulent Entamoeba histolytica strains identified by suppression subtractive hybridization.

Entamoeba histolytica is a parasite which presents capacity to degrade tissues and therefore has a pathogenic behavior. As this behavior is not shown by all strains, there have been several studies investigating molecular basis of the cytotoxicity process. Using the suppression subtractive hybridization (SSH) technique, differential gene expressions of two E. histolytica strains, one virulent (EGG) and one nonvirulent (452), have been analyzed with the purpose of isolating genes which may be involved with amoebic virulence. Nine cDNA fragments presenting high homology with E. histolytica previously sequenced genes were subtracted. Of these, four genes were confirmed by RT-PCR. Two coding for hypothetical proteins, one for a cysteine-rich protein, expressed only in the virulent strain, EGG and another one, coding for grainin 2 protein, exclusive from 452 strain. This study provided new insight into the proteins differences in the virulent and nonvirulent E. histolytica strains. We believe that further studies with these proteins may prove association of them with tissue damage, providing new perceptions to improve treatment or diagnosis of the invasive disease.

Complexes of different nitrogen donor heterocyclic ligands with SbCl3 and PhSbCl2 as potential antileishmanial agents against Sb(III)-sensitive and -resistant parasites.

Novel trivalent antimony complexes with the nitrogen donor heterocyclic ligand 2,2'-bipyridine (bipy), 1,10-phenanthroline (phen) or dipyrido[3,2-d:2',3'-f]quinoxaline (dpq) have been synthesized by the reaction with SbCl3 or PhSbCl2. The crystal structures of [Sb(phen)Cl3] and [PhSb(phen)Cl2]CH3COOH were determined and shown to adopt a distorted square pyramid geometry with a five-coordinated Sb center. Surprisingly, all the complexes, the ligands and PhSbCl2 showed very high antileishmanial activities, with IC50 in the nanomolar range against Sb(III)-sensitive and -resistant Leishmania infantum (syn. Leishmania chagasi) and Leishmania amazonensis strains. These compounds were much more active against these Leishmania strains than the old trivalent drug potassium antimonyl tartrate. [PhSb(phen)Cl2]CH3COOH complex was found to be the most active compound and the lack of cross-resistance of PhSbCl2 suggests that the transport pathways of this compound across the cell membrane differ from those responsible for the resistance of Leishmania to Sb(OH)3. In the case of the complexes with PhSbCl2, our data supports the model that both ligand and metal contributed to the overall activity of the complex. Furthermore, among the complexes with SbCl3, only bipy showed an improved activity upon complexation. Cytotoxicity evaluations of these compounds against murine peritoneal macrophages showed high selective indexes in the range of 7-70 for [Sb(phen)Cl3], [Sb(bipy)Cl3] and [Sb(dpq)Cl3] complexes, being much more selective than potassium antimonyl tartrate. In conclusion, this study presents a set of new antileishmanial agents including one of the most active Sb-based compounds ever reported, which can contribute to the development of new chemotherapeutic strategies against leishmaniasis including Sb-resistant cases.

Amphiphilic Antimony(V) Complexes for Oral Treatment of Visceral Leishmaniasis.

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.

Potent naphthoquinones against antimony-sensitive and -resistant Leishmania parasites: synthesis of novel α- and nor-α-lapachone-based 1,2,3-triazoles by copper-catalyzed azide-alkyne cycloaddition.

Continuing our screening program for novel anti-parasite compounds, we synthesized seven 1,4-naphthoquinones coupled to 1,2,3-triazoles, five nor-ß-lapachone-based 1,2,3-triazoles and ten α-lapachone-based 1,2,3-triazoles. These and other naphthoquinonoid compounds were evaluated for their activity against promastigote forms of antimony-sensitive and -resistant strains of Leishmania infantum (syn. Leishmania chagasi) and Leishmania amazonensis. The toxicity of these compounds to mammalian cells was also examined. The substances were more potent than an antimonial drug, with IC50 values ranging from 1.0 to 50.7 µM. Nor-α-lapachone derivatives showed the highest antileishmanial activity, with selectivity indices in the range of 10-15. These compounds emerged as important leads for further investigation as antileishmanial agents. Additionally, one of these compounds exhibited cross-resistance in Sb-resistant Leishmania and could provide a molecular tool for investigating the multidrug resistance mechanisms in Leishmania parasites.

An alternative in vitro drug screening test using Leishmania amazonensis transfected with red fluorescent protein.

Fluorescent and colorimetric reporter genes are valuable tools for drug screening models, since microscopy is labor intensive and subject to observer variation. In this work, we propose a fluorimetric method for drug screening using red fluorescent parasites. Fluorescent Leishmania amazonensis were developed after transfection with integration plasmids containing either red (RFP) or green fluorescent protein (GFP) genes. After transfection, wild-type (LaWT) and transfected (LaGFP and LaRFP) parasites were subjected to flow cytometry, macrophage infection, and tests of susceptibility to current antileishmanial agents and propranolol derivatives previously shown to be active against Trypanosoma cruzi. Flow cytometry analysis discriminated LaWT from LaRFP and LaGFP parasites, without affecting cell size or granulosity. With microscopy, transfection with antibiotic resistant genes was not shown to affect macrophage infectivity and susceptibility to amphotericin B and propranolol derivatives. Retention of fluorescence remained in the intracellular amastigotes in both LaGFP and LaRFP transfectants. However, detection of intracellular RFP parasites was only achieved in the fluorimeter. Murine BALB/c macrophages were infected with LaRFP parasites, exposed to standard (meglumine antimoniate, amphotericin B, Miltefosine, and allopurinol) and tested molecules. Although it was possible to determine IC(50) values for 4 propranolol derivatives (1, 2b, 3, and 4b), all compounds were considered inactive. This study is the first to develop a fluorimetric drug screening test for L. amazonensis RFP. The fluorimetric test was comparable to microscopy with the advantage of being faster and not requiring manual counting.

Improved antileishmanial activity of Dppz through complexation with antimony(III) and bismuth(III): investigation of the role of the metal.

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]∙H2O∙CH3OH 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.

Ecto-nucleotidase activities of promastigotes from Leishmania (Viannia) braziliensis relates to parasite infectivity and disease clinical outcome.

BACKGROUND: Leishmania (Viannia) braziliensis has been associated with a broad range of clinical manifestations ranging from a simple cutaneous ulcer to destructive mucosal lesions. Factors leading to this diversity of clinical presentations are not clear, but parasite factors have lately been recognized as important in determining disease progression. Given the fact that the activity of ecto-nucleotidases correlates with parasitism and the development of infection, we evaluated the activity of these enzymes in promastigotes from 23 L. braziliensis isolates as a possible parasite-related factor that could influence the clinical outcome of the disease. METHODOLOGY/PRINCIPAL FINDINGS: Our results show that the isolates differ in their ability to hydrolyze adenine nucleotides. Furthermore, we observed a positive correlation between the time for peak of lesion development in C57BL/6J mice and enzymatic activity and clinical manifestation of the isolate. In addition, we found that L. (V.) braziliensis isolates obtained from mucosal lesions hydrolyze higher amounts of adenine nucleotides than isolates obtained from skin lesions. One isolate with high (PPS6m) and another with low (SSF) ecto-nucleotidase activity were chosen for further studies. Mice inoculated with PPS6m show delayed lesion development and present larger parasite loads than animals inoculated with the SSF isolate. In addition, PPS6m modulates the host immune response by inhibiting dendritic cell activation and NO production by activated J774 macrophages. Finally, we observed that the amastigote forms from PPS6m and SSF isolates present low enzymatic activity that does not interfere with NO production and parasite survival in macrophages. CONCLUSIONS/SIGNIFICANCE: Our data suggest that ecto-nucleotidases present on the promastigote forms of the parasite may interfere with the establishment of the immune response with consequent impaired ability to control parasite dissemination and this may be an important factor in determining the clinical outcome of leishmaniasis.

Efficacy of combined therapy with liposome-encapsulated meglumine antimoniate and allopurinol in treatment of canine visceral leishmaniasis.

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.

Evaluation of parasitological and immunological parameters of Leishmania chagasi infection in BALB/c mice using different doses and routes of inoculation of parasites.

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.