Antileishmanial activity and mechanism of action from a purified fraction of Zingiber officinalis Roscoe against Leishmania amazonensis.

In recent years, considerable attention has been given to identify new antileishmanial products derived from medicinal plants, although, to date, no new effective compound has been recently applied to treat leishmaniasis. In the present study, the antileishmanial activity of a water extract from Zingiber officinalis Roscoe (ginger) was investigated and a purified fraction, named F10, was identified as responsible by this biological activity. The chemical characterization performed for this fraction showed that it is mainly composed by flavonoids and saponins. The water extract and the F10 fraction presented IC50 values of 125.5 and 49.8 µg/mL, respectively. Their selectivity indexes (SI) were calculated and values were seven and 40 times higher, respectively, in relation to the value found for amphotericin B, which was used as a control. Additional studies were performed to evaluate the toxicity of these compounds in human red blood cells, besides of the production of nitrite, as an indicator of nitric oxide (NO), in treated and infected macrophages. The results showed that both F10 fraction and water extract were not toxic to human cells, and they were able to stimulate the nitrite production, with values of 13.6 and 5.4 µM, respectively, suggesting that their biological activity could be due to macrophages activation via NO production. In conclusion, the present study shows that a purified fraction from ginger could be evaluated in future works as a therapeutic alternative, on its own or in association with other drugs, to treat disease caused by L. amazonensis.

Antileishmanial activity of compounds produced by endophytic fungi derived from medicinal plant Vernonia polyanthes and their potential as source of bioactive substances.

The purpose of this work was to evaluate the antileishmanial activity of endophytic fungi isolated from leaves of Vernonia polyanthes plant and their prospective use in the discovery of bioactive compounds. Sixteen endophytes were isolated by using potato dextrose agar medium and submitted to cultivation in rice medium. The fungal cultures were extracted with ethanol and used as crude extracts for testing their antileishmanial activity. The most active ethanol extract was obtained from P2-F3 strain, which was identified as Cochliobolus sativus by ITS rRNA gene sequence data. Followed by a bioassay-guided fractionation, the cochlioquinone A, isocochlioquinone A and anhydrocochlioquinone A compounds were isolated from the crude extracts and demonstrated to inhibit the parasites. From the present work, it is possible to conclude that endophytic fungi derived from medicinal plant V. polyanthes may be considered promising source for the discovery of bioactive compounds.

Antileishmanial activity and cytotoxicity of Brazilian plants.

Leishmaniasis is a major public health problem, and the alarming spread of parasite resistance has increased the importance of discovering new therapeutic products. The present study aimed to investigate the in vitro leishmanicidal activity from 16 different Brazilian medicinal plants. Stationary-phase promastigotes of Leishmania amazonensis and murine macrophages were exposed to 44 plant extracts or fractions for 48 h at 37°C, in order to evaluate their antileishmanial activity and cytotoxicity, respectively. The most potent extracts against L. amazonensis were the hexanic extract of Dipteryx alata (IC50 of 0.08 µg/mL), the hexanic extract of Syzygium cumini (IC50 of 31.64 µg/mL), the ethanolic and hexanic extracts of leaves of Hymenaea courbaril (IC50 of 44.10 µg/mL and 35.84 µg/mL, respectively), the ethanolic extract of H. stignocarpa (IC50 of 4.69 µg/mL), the ethanolic extract of Jacaranda caroba (IC50 of 13.22 µg/mL), and the ethanolic extract of J. cuspidifolia leaves (IC50 of 10.96 µg/mL). Extracts of D. alata and J. cuspidifolia presented higher selectivity index, with high leishmanicidal activity and low cytotoxicity in the mammalian cells. The capacity in treated infected macrophages using the extracts and/or fractions of D. alata and J. cuspidifolia was also analyzed, and reductions of 95.80%, 98.31%, and 97.16%, respectively, in the parasite burden, were observed. No nitric oxide (NO) production could be observed in the treated macrophages, after stimulation with the extracts and/or fractions of D. alata and J. cuspidifolia, suggesting that the biological activity could be due to mechanisms other than macrophage activation mediated by NO production. Based on phytochemistry studies, the classes of compounds that could contribute to the observed activities are also discussed. In conclusion, the data presented in this study indicated that traditional medicinal plant extracts present effective antileishmanial activity. Future studies could focus on the identification and purification of the antileishmanial compounds within these plants for analysis of their in vivo antileishmanial activity.

Propolis--based chitosan varnish: drug delivery, controlled release and antimicrobial activity against oral pathogen bacteria.

BACKGROUND: Dental caries is the most prevalent oral disease in several Asian and Latin American countries. It is an infectious disease and different types of bacteria are involved in the process. Synthetic antimicrobials are used against this disease; however, many of these substances cause unwarranted undesirable effects like vomiting, diarrhea and tooth staining. Propolis, a resinous substance collected by honeybees, has been used to control the oral microbiota. So, the objective of this study was to develop and characterize sustained-release propolis-based chitosan varnish useful on dental cariogenic biofilm prevention, besides the in vitro antimicrobial activity. METHODS: Three formulations of propolis - based chitosan varnish (PCV) containing different concentrations (5%, 10% and 15%) were produced by dissolution of propolis with chitosan on hydro-alcoholic vehicle. Bovine teeth were used for testing adhesion of coatings and to observe the controlled release of propolis associated with varnish. It was characterized by infrared spectroscopy, scanning electron microscopy, casting time, diffusion test in vitro antimicrobial activity and controlled release. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were tested for the main microorganisms involved in the cariogenic biofilm through the microdilution test in 96-well plates. RESULTS: The formulations presented a tooth surface adherence and were able to form films very fast on bovine tooth surface. Also, propolis-based chitosan varnishes have shown antimicrobial activity similar to or better than chlorhexidine varnish against all oral pathogen bacteria. All microorganisms were sensitive to propolis varnish and chitosan. MIC and MBC for microorganisms of cariogenic biofilme showed better results than chlorhexidine. Propolis active components were released for more than one week. CONCLUSION: All developed formulations turn them, 5%, 10% and 15% propolis content varnish, into products suitable for clinical application on dental caries prevention field, deserving clinical studies to confirm its in vivo activity.

Prophylactic or therapeutic administration of Agaricus blazei Murill is effective in treatment of murine visceral leishmaniasis.

The present study aimed to investigate the in vitro antileishmanial activity of five fractions obtained from Agaricus blazei water extract (AbM), namely, Fab1, Fab2, Fab3, Fab4, and Fab5; and use the selected leishmanicidal fraction to treat BALB/c mice infected with Leishmania chagasi. A curve dose-titration was performed to obtain the concentration to be test in infected animals. In this context, Fab5 fraction and AbM were used in the doses of 20 and 100 mg/kg/day, respectively, with the product been administered once a day. The effect induced by a chemo-prophylactic regimen, based on the administration Fab5 fraction and AbM 5 days before infection, and maintained for an additional 20 days post-infection was compared to a therapeutic regimen, in which the compounds were administered from 0 to 20 days of infection. Control animals were either treated with amphotericin B deoxycholate (AmpB) or received distilled water. All groups were followed up for 10 weeks post-infection, when parasitological and immunological parameters were analyzed. The Fab5 presented the best results of in vitro leishmanicidal activity. In the in vivo experiments, the use of Fab5 or AbM, as compared to control groups, resulted in significant reduced parasite burdens in the liver, spleen, and draining lymph nodes of the infected animals, as compared to control groups. A Type 1 immune response was observed in the Fab5 or AbM treated animals. No significant toxicity was observed. The chemo-prophylactic regimen proved to be more effective to induce theses responses. In this context, the data presented in this study showed the potential of the purified Fab5 fraction of AbM as a therapeutic alternative to treat visceral leishmaniasis. In addition, it can be postulated that this fraction can be also employed in a chemo-prophylactic regimen associated or not with other therapeutic products.

Therapeutic efficacy induced by the oral administration of Agaricus blazei Murill against Leishmania amazonensis.

The development of therapeutic alternatives to treat leishmaniasis has received considerable attention. The present study aimed to investigate the efficacy of the Agaricus blazei Murill water extract (AbM) to treat BALB/c mice infected with Leishmania amazonensis. First, a dose-titration curve was performed. The most well-defined concentration able to induce the most effective results in the infected animals, considering a daily administration of the product, was that of 100 mg kg(-1) day(-1). In this context, the AbM was administered orally, beginning on day 0 up to 20 days postinfection. Additional animals were treated with amphotericin B (AmpB, 5 mg kg(-1) day(-1)) by peritoneal route for the same period of time, while the control group received distilled water. The animals were evaluated at 14 weeks post-infection, at which time the parasitological and immunological parameters were analyzed. Mice treated with the AbM presented a 60% reduction in the inflammation of infected footpads as compared to untreated control-infected mice. Moreover, in the treated mice, as compared to the untreated controls, approximately 60 and 66% reductions could be observed in the parasite burdens of the footpad and draining lymph nodes, respectively. In addition, no parasites could be detected in the spleen of treated mice at week 14 postinfection. These treated animals produced significantly higher levels of interferon gamma (IFN-γ) and nitric oxide (NO), higher levels of parasite-specific IgG2a isotype antibodies, and lower levels of interleukin (IL)-4, and IL-10 in the spleen and lymph node cell cultures than did the controls. Differences could be observed by comparing animals treated with AbM to those treated with AmpB, as indicated by a significant reduction in tissue parasitism, higher levels of IFN-γ and NO, and lower levels of IL-4 and IL-10, as well as by a decreased hepatic toxicity. In conclusion, the present study's data show that the A. blazei Murill water extract presents a high potential for the treatment of leishmaniasis, although additional studies on mice, as well as on other mammal hosts, are warranted in an attempt to determine this extract's true efficacy as compared to other known therapeutic products.

Use of chitosan as pharmaceutical excipient in ocular drug delivery systems: Sterilization and pharmacokinetics.

The use of chitosan as a pharmaceutical excipient in the ocular field is already established. Nevertheless, some aspects related to its ocular administration, such as sterilization and excipient's pharmacokinetics, remain unclear. So, in this study, we evaluated those two relevant aspects, related to chitosan administration in eye. We used chitosan-based ocular inserts (CI) as formulation model. CI were produced by solvent/casting method and sterilized by saturated steam. Sterilization was confirmed by direct inoculation of inserts in suitable microbiological growth media. Physicochemical characterization of inserts before and after sterilization was performed. Results suggested that, although steam sterilization changed the arrangement of the matrix, the heat and the humidity did not modify the structure of the main polymeric chain. Pharmacokinetics of CI radiolabeled with technetium-99m (99m Tc) was assessed by scintigraphic images and ex vivo biodistribution study, after ocular administration in male Wistar rats. Scintigraphic and images analysis and ex vivo biodistribution study showed that the insert remained mainly in the eye until 6 hr after administration and its degradation products began to migrate to the abdominal cavity after 18 hr. Together, these data represent an important step forward the manufacturing and the clinical application of CI in the ophthalmic field.

Chitosan/hydroxyethyl cellulose inserts for sustained-release of dorzolamide for glaucoma treatment: In vitro and in vivo evaluation.

Eye drops containing hydrophilic drugs are commonly used to reduce intraocular pressure (IOP) in glaucoma patients, but compliance to the treatement is commonly reduced by frequent dosing and eventual systemic side effects. Sustained-release drug delivery systems, such as ocular inserts, can reduce dosing, limit systemic exposure, reduce side effects, and, then, improve patient adherence to therapy. Here, we developed and evaluated chitosan/hydroxyethyl cellulose-based ocular inserts for sustained release of dorzolamide, a hydrophilic drug. Dorzolamide inserts (DI) were produced by solvent/casting method and characterized by various physicochemical techniques. Pharmacokinetics studies were performed using scintigraphic images and ex vivo biodistribution. The effectiveness of inserts was tested in glaucomatous rats. Characterization studies showed that the drug strongly interacted with the polymeric matrix, but in vitro results showed that DI took only 3 h to release 75% of dorzolamide entraped. However, scintigraphic images and ex vivo biodistribution studies revealed that more than 50% of 99mTc-dorzolamide remained in the eye after 18 h of DI administration, while only about 30% of the drug remained in the eye after drops instilation. DI exerted significant hypotensive effect for two weeks, after single administration, while IOP values remained high in placebo and untreated groups. Eye drops were effective only during the treatment period. Only DI treatment prevented retinal ganglion cells death. Altogether, these findings evidenced the potential application of polymeric-based inserts for sustained release of dorzolamide in glaucoma management.

Scintigraphic imaging using technetium-99m-labeled ceftizoxime in an experimental model of acute osteomyelitis in rats.

PURPOSE: To investigate inflammatory (zymosan) and infectious (Staphylococcus aureus) processes in experimental models in rats using technetium-99m-labeled ceftizoxime (CFT). METHODS: Male Wistar rats were used for the development of the inflammatory (zymosan) and infectious (S. aureus) processes in the medullary cavity of the left tibia. Sterile saline was used for the control group. At 48 h after induction of the processes, the animals were anesthetized and scintigraphic images were acquired at 1, 2, 4, and 6 h after intravenous injection of 0.1 ml of 99mTc-CFT (55 MBq). Quantitative analysis of the scintigraphic images was performed by counting the radioactivity in the regions of interest. Samples of tibia were taken for histopathological examination. RESULTS: The images showed that 99mTc-CFT presented higher tropism to infectious foci than with the inflammatory site. The average value of the target/nontarget ratio of the 99mTc-CFT was significantly higher in the infected (2.40+/-0.22) than in the inflamed tibia (1.50+/-0.05) and the control group (1.05+/-0.04) for all of the investigated times. The histological data showed a similar inflammatory response for both the S. aureus and zymosan groups. CONCLUSION: The 99mTc-CFT presented a high tropism and retention for an infected region in this model of osteomyelitis, thereby constituting an interesting strategy to distinguish aseptic from septic sites.

Antileishmanial activity of standardized fractions of Stryphnodendron obovatum (Barbatimão) extract and constituent compounds.

ETHNOPHARMACOLOGICAL RELEVANCE: Stryphnodendron obovatum Benth. is a Brazilian tree used to treat skin ulceration, promote wound healing, and inhibit the growth of protozoa, including Trypanosoma and Leishmania species. Bioguided fractionation of the ethanol extract of S. obovatum stem bark was performed, and antileishmanial and antioxidant activities of the standardized fractions were analyzed. MATERIALS AND METHODS: Stationary-phase Leishmania amazonensis promastigotes, murine macrophages, and human red blood cells (RBCs) were exposed to plant extract, standardized fractions or isolated compounds for 48 h at 37 °C to evaluate their antiparasitic activity and cytotoxicity. The 2,2-diphenyl-1-picryl-hidrazyl assay was used to evaluate antioxidant activity. RESULTS: The S. obovatum extract and fractions showed antileishmanial and antioxidant activity; however, the organic fraction (OF) showed the best efficacy. We identified gallic acid, gallocatechin, epigallocatechin, catechin, and epigallocatechin gallate in the OF fraction. These compounds effectively inhibited L. amazonensis activity, with gallic acid, gallocatechin, and epigallocatechin gallate showing the highest selectivity. Furthermore, the evaluated compounds had no significant effect on murine macrophages and human RBCs. CONCLUSIONS: The compounds present in the S. obovatum plant bark ethanol extract may provide an alternative therapeutic approach for L. amazonensis treatment.

Synthesis and evaluation of the antiparasitic activity of bis-(arylmethylidene) cycloalkanones.

A series of bis-(arylmethylidene)-cycloalkanones was synthesized by cross-aldol condensation. The activity of the compounds was evaluated against amastigotes forms of Trypanosoma cruzi and promastigotes forms of Leishmania amazonensis. The cytotoxicity of the active compounds on uninfected fibroblasts or macrophages was established in vitro to evaluate the selectivity of their antiparasitic effects. Six compounds displayed trypanocidal activity against amastigotes intracellular forms of T. cruzi with IC50 values ranging from 7.0 to 249 µM. Besides these six compounds, eight other molecules exhibited significant leishmanicidal activity (IC50 values ranging from 0.6 to 110.4 µM). Two compounds can be considered as promising antiparasitic lead molecules because they showed IC50 values in the low-micromolar range (≤1.2 µM) with an adequate SI (≥19.9). To understand the mechanism of action of these compounds, two possible molecular targets were investigated: trypanothione reductase (TR) and cruzain.

An optimized nanoparticle delivery system based on chitosan and chondroitin sulfate molecules reduces the toxicity of amphotericin B and is effective in treating tegumentary leishmaniasis.

Amphotericin B (AmpB) is active against leishmaniasis, but its use is hampered due to its high toxicity observed in patients. In this study, a nanoparticles-delivery system for AmpB (NQC-AmpB), containing chitosan and chondroitin sulfate molecules, was evaluated in BALB/c mice against Leishmania amazonensis. An in vivo biodistribution study, including biochemical and toxicological evaluations, was performed to evaluate the toxicity of AmpB. Nanoparticles were radiolabeled with technetium-99m and injected in mice. The products presented a similar biodistribution in the liver, spleen, and kidneys of the animals. Free AmpB induced alterations in the body weight of the mice, which, in the biochemical analysis, indicated hepatic and renal injury, as well as morphological damage to the kidneys of the animals. In general, no significant organic alteration was observed in the animals treated with NQC-AmpB. Mice were infected with L. amazonensis and treated with the nanoparticles or free AmpB; then, parasitological and immunological analyses were performed. The NQC-AmpB group, as compared to the control groups, presented significant reductions in the lesion size and in the parasite burden in all evaluated organs. These animals presented significantly higher levels of IFN-γ and IL-12, and low levels of IL-4 and IL-10, when compared to the control groups. The NQC-AmpB system was effective in reducing the infection in the animals, and proved to be effective in diminishing the toxicity evoked by AmpB, which was observed when it was administered alone. In conclusion, NQC-AmpB could be considered a viable possibility for future studies in the treatment of leishmaniasis.

Subtractive phage display selection from canine visceral leishmaniasis identifies novel epitopes that mimic Leishmania infantum antigens with potential serodiagnosis applications.

Visceral leishmaniasis (VL) is a zoonotic disease that is endemic to Brazil, where dogs are the main domestic parasite reservoirs, and the percentages of infected dogs living in regions where canine VL (CVL) is endemic have ranged from 10% to 62%. Despite technological advances, some problems have been reported with CVL serodiagnosis. The present study describes a sequential subtractive selection through phage display technology from polyclonal antibodies of negative and positive sera that resulted in the identification of potential bacteriophage-fused peptides that were highly sensitive and specific to antibodies of CVL. A negative selection was performed in which phage clones were adhered to purified IgGs from healthy and Trypanosoma cruzi-infected dogs to eliminate cross-reactive phages. The remaining supernatant nonadhered phages were submitted to positive selection against IgG from the blood serum of dogs that were infected with Leishmania infantum. Phage clones that adhered to purified IgGs from the CVL-infected serum samples were selected. Eighteen clones were identified and their reactivities tested by a phage enzyme-linked immunosorbent assay (phage-ELISA) against the serum samples from infected dogs (n = 31) compared to those from vaccinated dogs (n = 21), experimentally infected dogs with cross-reactive parasites (n = 23), and healthy controls (n = 17). Eight clones presented sensitivity, specificity, and positive and negative predictive values of 100%, and they showed no cross-reactivity with T. cruzi- or Ehrlichia canis-infected dogs or with dogs vaccinated with two different commercial CVL vaccines in Brazil. Our study identified eight mimotopes of L. infantum antigens with 100% accuracy for CVL serodiagnosis. The use of these mimotopes by phage-ELISA proved to be an excellent assay that was reproducible, simple, fast, and inexpensive, and it can be applied in CVL-monitoring programs.

Novel targeting using nanoparticles: an approach to the development of an effective anti-leishmanial drug-delivery system.

The study reported here aimed to develop an optimized nanoparticle delivery system for amphotericin B (AmpB) using a polyelectrolyte complexation technique. For this, two oppositely charged polymers presenting anti-leishmanial activity - chitosan (Cs) and chondroitin sulfate (ChS) - were used: Cs as a positively charged polymer and ChS as a negatively charged polymer. The chitosan (NQ) nanoparticles, chitosan-chondroitin sulfate (NQC) nanoparticles, and chitosan-chondroitin sulfate-amphotericin B (NQC-AmpB) nanoparticles presented a mean particle size of 79, 104, and 136 nm, respectively; and a polydispersity index of 0.2. The measured zeta potential of the nanoparticles indicated a positive charge in their surface, while scanning and transmission electron microscopy revealed spherical nanoparticles with a smooth surface. Attenuated total reflectance-Fourier transform infrared spectroscopy analysis showed an electrostatic interaction between the polymers, whereas the release profile of AmpB from the NQC-AmpB nanoparticles showed a controlled release. In addition, the Cs; ChS; and NQ, NQC, and NQC-AmpB nanoparticles proved to be effective against promastigotes of Leishmania amazonensis and Leishmania chagasi, with a synergistic effect observed between Cs and ChS. Moreover, the applied NQ, NQC, and NQC-AmpB compounds demonstrated low toxicity in murine macrophages, as well as null hemolytic activity in type O(+) human red blood cells. Pure AmpB demonstrated high toxicity in the macrophages. The results show that cells infected with L. amazonensis and later treated with Cs, ChS, NQ, NQC, NQC-AmpB nanoparticles, or pure AmpB presented with a significant reduction in parasite number in the order of 24%, 31%, 55%, 66%, 90%, and 89%, respectively. The data presented indicate that the engineered NQC-AmpB nanoparticles could potentially be used as an alternative therapy to treat leishmaniasis, mainly due its low toxicity to mammals' cells.

Correction: Antigenicity and Protective Efficacy of a Leishmania Amastigote-specific Protein, Member of the Super-oxygenase Family, against Visceral Leishmaniasis.

[This corrects the article on p. e2148 in vol. 7.].

Sensitive and specific serodiagnosis of Leishmania infantum infection in dogs by using peptides selected from hypothetical proteins identified by an immunoproteomic approach.

In Brazil, the percentage of infected dogs living in areas where canine visceral leishmaniasis (CVL) is endemic ranges from 10 to 62%; however, the prevalence of infection in dogs is probably higher than figures reported from serological studies. In addition, problems with the occurrence of false-positive or false-negative results in the serodiagnosis of CVL have been reported. The present work analyzed the potential of synthetic peptides mapped from hypothetical proteins for improvement of the serodiagnosis of Leishmania infantum infection in dogs. From 26 identified leishmanial proteins, eight were selected, considering that no homologies between these proteins and others from trypanosomatide sequence databases were encountered. The sequences of these proteins were mapped to identify linear B-cell epitopes, and 17 peptides were synthesized and tested in enzyme-linked immunosorbent assays (ELISAs) for the serodiagnosis of L. infantum infection in dogs. Of these, three exhibited sensitivity and specificity values higher than 75% and 90%, respectively, to differentiate L. infantum-infected animals from Trypanosoma cruzi-infected animals and healthy animals. Soluble Leishmania antigen (SLA) showed poor sensitivity (4%) and specificity (36%) to differentiate L. infantum-infected dogs from healthy and T. cruzi-infected dogs. Lastly, the three selected peptides were combined in different mixtures and higher sensitivity and specificity values were obtained, even when sera from T. cruzi-infected dogs were used. The study's findings suggest that these three peptides can constitute a potential tool for more sensitive and specific serodiagnosis of L. infantum infection in dogs.

Antigenicity and protective efficacy of a Leishmania amastigote-specific protein, member of the super-oxygenase family, against visceral leishmaniasis.

BACKGROUND: The present study aimed to evaluate a hypothetical Leishmania amastigote-specific protein (LiHyp1), previously identified by an immunoproteomic approach performed in Leishmania infantum, which showed homology to the super-oxygenase gene family, attempting to select a new candidate antigen for specific serodiagnosis, as well as to compose a vaccine against VL. METHODOLOGY/PRINCIPAL FINDINGS: The LiHyp1 DNA sequence was cloned; the recombinant protein (rLiHyp1) was purified and evaluated for its antigenicity and immunogenicity. The rLiHyp1 protein was recognized by antibodies from sera of asymptomatic and symptomatic animals with canine visceral leishmaniasis (CVL), but presented no cross-reactivity with sera of dogs vaccinated with Leish-Tec, a Brazilian commercial vaccine; with Chagas' disease or healthy animals. In addition, the immunogenicity and protective efficacy of rLiHyp1 plus saponin was evaluated in BALB/c mice challenged subcutaneously with virulent L. infantum promastigotes. rLiHyp1 plus saponin vaccinated mice showed a high and specific production of IFN-γ, IL-12, and GM-CSF after in vitro stimulation with the recombinant protein. Immunized and infected mice, as compared to the control groups (saline and saponin), showed significant reductions in the number of parasites found in the liver, spleen, bone marrow, and in the paws' draining lymph nodes. Protection was associated with an IL-12-dependent production of IFN-γ, produced mainly by CD4 T cells. In these mice, a decrease in the parasite-mediated IL-4 and IL-10 response could also be observed. CONCLUSIONS/SIGNIFICANCE: The present study showed that this Leishmania oxygenase amastigote-specific protein can be used for a more sensitive and specific serodiagnosis of asymptomatic and symptomatic CVL and, when combined with a Th1-type adjuvant, can also be employ as a candidate antigen to develop vaccines against VL.