Utility of the combination of hederagenin glucoside saponins and chromane hydrazone in the topical treatment of canine cutaneous leishmaniasis. An observational study.

Canine cutaneous leishmaniasis (CCL) is an emerging zoonotic infection endemic in several countries of the world. Due to variable response to therapy and frequency of relapses, a more effective, safer, and inexpensive treatment is needed. Recently, it was reported that the hederagenin glucoside saponins (SS) and chromane-derived hydrazone (TC2) combined in a 1:1 ratio has high potential in antileishmanial therapy since both compounds alter the survival of Leishmania and the ability to infect adjacent macrophage. Not only the skin permeation and the absorption of an ointment containing 2% TC2 and 2% SS (w/w) was determined in this work, but also the acute dermal toxicity in both in vitro and in vivo assays. Last, the effectiveness and safety of the topical therapy with 2% TC2-2% SS ointment was evaluated in an observational study in dogs with diagnosis of cutaneous leishmaniasis (CL). Both TC2 and SS diffused through pig ear skin and traces of TC2 (but not SS) were detected in the stratum corneum of mice at 6-24 h. Neither TC2 nor SS was detected in plasma. The acute dermal toxicity was negative. Treatment with 2% TC2-2% SS ointment produced a complete long-term clinical cure in 56 dogs (24 females and 32 males) from the Orinoco and Amazonas regions in southeastern Colombia without adverse effects. All dogs have remained disease-free for the last 24 months. In conclusion, these results support the use of this topical therapy as a safer and new first-line local treatment of CCL that could help limit the spread of CL from dogs to humans.

Preparation and in vitro evaluation of PLA/biphasic calcium phosphate filaments used for fused deposition modelling of scaffolds.

Ceramic materials such as calcium phosphates (CaPs) with a composition similar to the mineral phase of bones and polymeric polylactic acid (PLA) are potential candidates for the manufacturing of scaffolds to act as bone substitutes and for tissue engineering applications, due to their bioresorbability and biocompatibility. Variables such as porosity, topography, morphology, and mechanical properties play an essential role in the scaffolds response. In this paper, a polymer/ceramic composite filament of 1.7 mm in diameter based on PLA and biphasic calcium phosphates (BCPs) was obtained by hot-melt extrusion in a single screw extruder. The particles of BCP were obtained by solution-combustion synthesis, and the PLA used was commercial grade. The BCPs ceramics were characterized by X-ray diffraction (XRD), scanning electron microscopic (SEM), transmission electron microscopy (TEM), and Brunauer, Emmett, and Teller (BET). It was possible to confirm that the main inorganic phases were hydroxyapatite (HAP) and tricalcium phosphate (TCP) with grain sizes below 100 nm and with high porosity. The Filaments obtained are a bit fragile but were able to be used in fused deposition modelling (FDM) using low-cost commercial printers. The filaments were characterized by SEM and energy dispersive X-ray (EDX). The in-vitro tests of filaments showed deposition of apatite phases on their surface, non-cytotoxic behavior, adequate cell proliferation and cell adhesion.

Synthesis of calcium phosphate nanostructures by combustion in solution as a potential encapsulant system of drugs with photodynamic properties for the treatment of cutaneous leishmaniasis.

The traditional drugs used in the treatment of cutaneous leishmanisis (CL) have multiple disadvantages, such as high toxicity, high costs, and more recently the appearance of parasites resistant to those drugs. For this reason, some research has focused on the development of new drugs or treatment therapies. Photodynamic therapy (PDT) that involves the use of a photosensitive or photosensitizing compound capable of producing reactive oxygen species to which Leishmania parasites are sensitive, has emerged as an alternative for the treatment of CL. However, some of these sensitizing compounds exhibit some toxicity (cytotoxicity, allergic reaction, etc), low selectivity, and some of them are insoluble in aqueous media limiting their use. Therefore, the PDT can be improved using encapsulation systems which protect drugs, prevent their degradation, help them overcome physical barriers and increase their selectivity. In this study, we propose the use of calcium phosphate as a potential encapsulant or photodynamic support for photoactive drugs, using hypericin (HY) as a photosensitizer agent. The self-combustion route was used to synthesize the CP nanostructures. The structure and morphology of CP nanoparticles were evaluated via X-ray diffraction (XRD), Raman and field-emission scanning electron microscopy (FE-SEM). Phases rich in hydroxyapatite and CP ß phase, with granular morphology and an average grain size of 42.9 nm were obtained. The encapsulation uptake and the interactions between HY and the encapsulated system were evaluated by fluorescence spectroscopy and Fourier-transform infrared spectroscopy (FTIR), respectively. Approximately 13% of HY was enapsulated per 1 µg of nanoparticles of calcium phosphate. Composites were submitted to in vitro assays of cytotoxicity and anti-leishmanial activity. The CP nanoparticles did not affect the photodynamic activity of HY. On the contrary they showed antileishmanial response.

Endemically exposed asymptomatic individuals show no increase in the specific Leishmania (Viannia) panamensis-Th1 immune response in comparison to patients with localized cutaneous leishmaniasis.

In Colombia, most cases of human cutaneous leishmaniasis are caused by Leishmania (Viannia) panamensis. Interestingly, up to 30% of the exposed population do not suffer from clinical leishmaniasis although it is likely that they are continuously infected with Leishmania parasites. Since it is believed that the induction of efficient Th1 immune responses protects against Leishmania infections both in humans and in animal models, we determined if endemically exposed asymptomatics showed stronger Leishmania-specific Th1 immune responses than patients with active localized cutaneous leishmaniasis (LCL). We found that Montenegro skin test responses were slightly higher among asymptomatic individuals compared to patients suffering from LCL. However, PBMC from patients with LCL showed similar Leishmania-specific proliferative responses compared to PBMC from asymptomatic individuals. Furthermore, PBMC from both groups also secreted similar amounts of IFN-gamma, IL-12p40 and IL-10 after in vitro exposure to L. panamensis. No IL-4 was detected in the supernatants. Taken together our results suggest that lack of LCL development in endemically exposed asymptomatics cannot be explained by stronger systemic anti-Leishmania Th1 immune responses or decreased Th2 responses in these individuals in comparison to individuals who develop LCL. It may be possible that other mechanisms are responsible for resistance to cutaneous leishmaniasis in Colombia in endemically exposed asymptomatics.

[Gender and cutaneous leishmaniasis in Colombia]. / Leishmaniosis cutánea en Colombia y género.

Leishmaniasis in Colombia has traditionally been seen as a health risk for adult males, as they become infected when they enter the vector's biotopes to tap natural resources. National health statistics seem to confirm this theory. However, during field studies, the Program for the Study and Control of Tropical Diseases (PECET) observed both equal proportions of men and women with active leishmaniasis and delayed hypersensitivity skin tests and equal proportions of males and females having had contact with the parasite from early childhood. Several factors that have not been analyzed in depth in Colombia thus far appear to distort the disease's epidemiological pattern in the country, and gender-linked differences in access to health care appear to exist. As a consequence, no relief is provided for this source of human suffering, and socioeconomic repercussions for households are significant. Preventive measures by the Colombian Ministry of Health (MOH) systematically underestimate the magnitude of intra- and peridomiciliary transmission, and female patients are excluded from active case detection. Further research should be devoted to this phenomenon. The MOH should be encouraged to improve leishmaniasis control programs, especially with regard to active case detection, training, and teaching, so that quicker diagnosis can be performed. Meanwhile, the MOH should retrain its health personnel.

Cytotoxicity and antileishmanial activity of Annona muricata pericarp.

Hexane, ethyl acetate and methanol extracts of Annona muricata pericarp were tested in vitro against Leishmania braziliensis and L. panamensis promastigotes, and against cell line U-937. The ethyl acetate extract was more active than the other extracts and even of Glucantime used as reference substance. Its fractionation led to the isolation of three acetogenins--annonacin, annonacin A and annomuricin A.

Epidemiology of paragonimiasis in Colombia.

Five newly discovered endemic foci for paragonimiasis in Colombia are described for the first time. The disease was diagnosed in 24 people from the Embera Indian communities located at the Colombian Pacific Coast and investigated in 1993-98. We also describe the clinical, epidemiological and treatment response aspects. In these foci an Aroapyrgus sp. snail different from A. colombiensis was found to be the first intermediate host, and the crab Hypolobocera emberarum nsp. the second intermediate host.

Safety and immunogenicity of a killed Leishmania (L.) amazonensis vaccine against cutaneous leishmaniasis in Colombia: a randomized controlled trial.

The safety and immunogenicity of an intramuscular (i.m.) and intradermal (ID) formulation of autoclaved Leishmania (Leishmania) amazonensis vaccine was evaluated in 296 volunteers in a randomized, placebo-controlled, double-blind trial in Colombia. There were 4 vaccination groups: i.m. vaccine, i.m. placebo, ID vaccine, and ID placebo. The ID formulations were mixed with BCG as adjuvant at the time of injection. For each group, 3 vaccinations were given with a 20-day interval between injections, and adverse events were monitored at 20 min, and at 2, 7 and 21 days after each injection. BCG-induced adverse reactions resulted in cancellation of the third vaccine administration in the ID groups. Antibody titres did not differ significantly between the groups. Montenegro skin-test conversion was achieved by 86.4% and 90% of the i.m. vaccine group and by 25% and 5% of the i.m. placebo group 80 days and 1 year after vaccination, respectively. A significant increase in mean Leishmania-antigen lymphocyte proliferation indexes was observed after i.m. vaccine immunization, but not after i.m. placebo immunization, 80 days and 1 year after vaccination. Significant levels of IFN gamma but not IL-10 were observed 1 year after vaccination in the i.m. vaccine group compared to the i.m. placebo group. The good safety profile and evidence of Th1 immune reactions due to i.m. vaccination in this phase-I/II study suggest that a population-based phase-III efficacy trial of the i.m. vaccine should be initiated.

Sensitivity to Glucantime of Leishmania viannia isolated from patients prior to treatment.

In vitro sensitivity to pentavalent antimony (SbV) as meglumine antimoniate (Glucantime) of Leishmania of the Viannia subgenus isolated prior to treatment from patients with uncomplicated cutaneous leishmaniasis was evaluated for intracellular amastigotes in the U-937 human monocytic cell line and log phase promastigotes. The 50% effective dose (ED50) of pharmaceutical and additive-free formulations of Glucantime were determined based on the kinetics of the response of Leishmania Viannia to SbV in vitro. ED50 to SbV was inversely related to time of exposure to drug. The potency of the additive-free formulation of Glucantime was significantly greater than that of the pharmaceutical formulation, irrespective of the parasite form. In vitro sensitivity to SbV ranged from < 5.3 micrograms/ml to > 170.0 micrograms/ml. Under the conditions used, 11 (39%) of 28 strains were sensitive to clinically achievable serum concentrations of SbV. No correlation was observed between the total amount of SbV required for healing of lesions and the in vitro response to the pharmaceutical formulation of Glucantime. In contrast, a significant correlation (P = 0.001) was observed between clinical response and the in vitro sensitivity of promastigotes to the additive-free formulation of Glucantime. The greater potency of the additive-free formulation of Glucantime, the correlation of in vitro sensitivity of promastigotes to this formulation and the clinical response to treatment, and the effect of time of exposure to SbV demonstrate the importance of assay conditions on the outcome and interpretation of in vitro evaluation of drug sensitivity.