Leishmanicidal potentials of Gossypium hirsutum extract and its fractions on Leishmania major in a murine model: parasite burden, gene expression, and histopathological profile.

Introduction. Leishmaniasis is a neglected tropical and subtropical disease caused by over 20 protozoan species.Hypothesis. Treatment of this complex disease with traditional synthetic drugs is a major challenge worldwide. Natural constituents are unique candidates for future therapeutic development.Aim. This study aimed to assess the in vivo anti-leishmanial effect of the Gossypium hirsutum extract, and its fractions compared to the standard drug (Glucantime, MA) in a murine model and explore the mechanism of action.Methodology. Footpads of BALB/c mice were infected with stationary phase promastigotes and treated topically and intraperitoneally with G. hirsutum extract, its fractions, or Glucantime, 4 weeks post-infection. The extract and fractions were prepared using the Soxhlet apparatus with chloroform followed by the column procedure.Results. The crude extract significantly decreased the footpad parasite load and lesion size compared to the untreated control group (P<0.05), as revealed by dilution assay, quantitative real-time PCR, and histopathological analyses. The primary mode of action involved an immunomodulatory role towards the Th1 response in the up-regulation of IFN-γ and IL-12 and the suppression of IL-10 gene expression profiling against cutaneous leishmaniasis caused by Leishmania major.Conclusion. This finding suggests that the extract possesses multiple combinatory effects of diverse bioactive phytochemical compositions that exert its mechanisms of action through agonistic-synergistic interactions. The topical extract formulation could be a suitable and unique candidate for future investigation and pharmacological development. Further studies are crucial to evaluate the therapeutic potentials of the extract alone and in combination with conventional drugs using clinical settings.

Efficacy of nanoemulsion with Pterodon emarginatus Vogel oleoresin for topical treatment of cutaneous leishmaniasis.

Cutaneous leishmaniasis (CL) is a neglected tropical skin disease caused by the protozoan genus Leishmania. The treatment is restricted to a handful number of drugs that exhibit toxic effects, limited efficacy, and drug resistance. Additionally, developing an effective topical treatment is still an enormous unmet medical challenge. Natural oils, e.g. the oleoresin from P. emarginatus fruits (SO), contain various bioactive molecules, especially terpenoid compounds such as diterpenes and sesquiterpenes. However, its use in topical formulations can be impaired due to the natural barrier of the skin for low water solubility compounds. Nanoemulsions (NE) are drug delivery systems able to increase penetration of lipophilic compounds throughout the skin, improving their topical effect. In this context, we propose the use of SO-containing NE (SO-NE) for CL treatment. The SO-NE was produced by a low energy method and presented suitable physicochemical characteristic: average diameter and polydispersity index lower than 180 nm and 0.2, respectively. Leishmania (Leishmania) amazonensis-infected BALB/c mice were given topical doses of SO or SO-NE. The topical use of a combination of SO-NE and intraperitoneal meglumine antimoniate reduced lesion size by 41 % and tissue regeneration was proven by histopathological analyses. In addition, a reduction in the parasitic load and decreased in the level of IFN-γ in the lesion may be associated, as well as a lower level of the cytokine IL-10 may be associated with a less intense inflammatory process. The present study suggests that SO-NE in combination meglumine antimoniate represents a promising alternative for the topical treatment of CL caused by L. (L.) amazonensis.

Role of magnetic Field in the Healing of Cutaneous Leishmaniasis Lesions in Mice.

Leishmaniasisis an important tropical cutaneous disease that is endemic in the Middle East, including Iran. There is no consensus on the appropriate therapeutic method, dose, and duration for this disease. The pentavalent antimonial compounds are the first-line treatments of leishmaniasis. With regard to the resistance of this disease against drugs and its treatment failure in some patients, the present study was conducted to investigate the role of alternating magnetic field (AMF) in the treatment of cutaneous leishmaniasis lesionsin mice. To this end, 45 male Balb/c mice with the age of 3-4 weeks and weight of 18-20 g were purchased from the Pasteur Institute, Tehran, Iran, to be investigated. The mice were infected with Leishmania promastigote (2&times;106) injected in the upper end of the tail.After 3 weeks, the animals were screened for developing lesions. Finally, 15 mice were selected and randomly divided into three equal groups of positive control (treated with the standard drug), negative control (without treatment), and experimental (subjected to AMF at a frequency of 50 kHz for 30 min on a daily basis for 4 weeks). The subjects were followed up for 4 weeks, and the size of their lesions was measured weekly. The data were analyzed by repeated measures test in SPSS software (version 20) at a p-value of &lt; 0.05. There was no significant difference between the experimental and positive control groups (P&gt;0.05). However, the negative control group showed a significant difference with the positive control and experimental groups (P=0.0001). As the findings indicated, AMF was seemingly able to decrease the size of lesions to the same extent as the standard drug. Consequently, AMF could be suggested as a noninvasive and complementary tool against cutaneous leishmaniasis. However, it is required to perform more studies to address different aspects of this domain.

In-vitro and in-vivo comparative effects of the spring and autumn-harvested Artemisia aucheri Bioss extracts on Leishmania major.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia aucheri Bioss contains flavonoid, coumarin and santonin with antioxidant, antimicrobial and antileishmanial effects. The current study was aimed to comparatively evaluate the effects of spring and autumn extracts of A. aucheri Bioss on Leishmania major both in-vitro and in-vivo conditions. METHODS: HPLC analysis was used to evaluate the percentages of compounds in spring and autumn extracts of A. aucheri. For in-vitro assay, the effect of different concentrations of spring and autumn extracts of A. aucheri was tested on L. major promastigotes and amastigotes. MTT and flow cytometry methods were used to evaluate the cytotoxicity and probable apoptosis of A. aucheri extracts on L. major promastigotes. On the other hand, for in-vivo assay, the extracts were used as ointments to treat lesions developed on BALB/c mice after 28 days post inoculation of L. major. The diameter of lesions and the survival rates of infected BALB/c mice were measured weekly for a period of two months. RESULTS: The HPLC analysis showed the substance Quercitrin was present in the spring A. aucheri extract but not in the autumn extract. The mean numbers of amastigotes in each treated macrophage with the spring and autumn A. aucheri extracts were 1.2 and 1.8 respectively, which showed statistically significant differences (P < 0.05). Flow cytometry revealed that the spring and autumn A. aucheri extracts caused about 32% and 3.78% apoptosis respectively. The inhibitory concentration (IC50) of spring and autumn A. aucheri extracts to amastigotes were determined to be 90 µg/mL and 183 µg/mL respectiovely. In-vivo, the diameter of lesions treated with the spring A. aucheri extract was significantly less (P < 0.05) compared to those treated with the autumn extract (2.6 and 7.8 mm respectively). Also, mice treated with spring A. aucheri extract had higher survival rates compared to control group. CONCLUSION: Given the above results, it can be concluded that spring A. aucheri extract has a greater fatality effect on L. major promastigotes in-vitro compared to the autum extract. In addition, the spring extract has stronger therapeutic effect on lesions caused by L. major in BALB/c mice than the autum extract.

N-acetyl-L-cysteine reduces Leishmania amazonensis-induced inflammation in BALB/c mice.

BACKGROUND: Leishmaniasis is a emergent disease characterized by different clinical manifestations in both humans and dogs. Predominant clinical features of cutaneous leishmaniasis are ulcerative painless skin lesions. Several data reported that pain is associated with human and dog leishmaniasis, out with areas of painless ulcerative lesions per se. Actually, current medications used for leishmaniasis management are characterized by several side effects and, in addition, some cases of the disease are refractory to the treatment. On this background it is mandatory the identification of new and safe candidates for designing less toxic and low-cost remedies. Therefore, the search for new leishmanicidal compounds is indispensable. METHODS: In the present paper we investigated the effect of orally N-acetyl-L-cysteine (NAC) supplementation at dose of 200 mg/Kg for 10 weeks, in subcutaneous Leishmania (L). amazonensis infected BALB/c mice. And evaluating the effect of NAC on inflammatory response such as TNF-α, IL-6, IL-1ß levels, and on thermal and mechanical hyperalgesia. RESULTS: In the present paper we showed how NAC supplementation affected parameters of oxidative stress (GSH, MDA, SOD), inflammation such as cytokines levels (IL-1ß, IL-6, TNFα) and mast cell activation and consequently on induced pain, during leishmaniosis in BALB\c mice. CONCLUSIONS: The findings of our study provided the scientific data demonstrating that L. amazonensis infection induces inflammation and pain in BALB/c mice that are reversed by administration of NAC.

Treatment of cutaneous leishmaniasis with thermotherapy in Brazil: an efficacy and safety study.

BACKGROUND: Pentavalent antimonials remain as the standard drugs in the treatment of cutaneous leishmaniosis. The high cost, difficult administration, long treatment time, toxicity and increasing morbidity are factors that limit the use of these drugs. OBJECTIVES: To describe the response to radiofrequency thermotherapy in the treatment of localized cutaneous leishmaniasis in Brazil, and to evaluate its safety and tolerability. METHODS: We conducted a non-comparative open trial with a total of 15 patients confirmed to have cutaneous leishmaniasis on parasitological examination. A single radiofrequency thermotherapy session at 50ºC for 30 seconds was applied to the lesion and its edges. In patients with more than one lesion, only the largest one was treated initially. If after 30 days there was no evidence of healing, the smaller lesion was also treated with thermotherapy. Clinical cure was defined as visible healing for three months after treatment. The patients were followed-up for six months and there was no follow-up loss. RESULTS: Of all 23 lesions, only two evolved to complete healing without the need of treatment. Of 21 lesions, 18 (85.7%) achieved full healing. The main observed side effects were itching, burning sensation, pain and blisters. STUDY LIMITATIONS: Sample with a small number of patients and short follow-up. CONCLUSION: Thermotherapy can be considered a therapeutic alternative in localized cutaneous leishmaniasis, especially in cases of single cutaneous lesions and with formal contraindications to conventional treatment with pentavalent antimonials.

Treatment of cutaneous leishmaniasis with thermotherapy in Brazil: an efficacy and safety study

Abstract: BACKGROUND: Pentavalent antimonials remain as the standard drugs in the treatment of cutaneous leishmaniosis. The high cost, difficult administration, long treatment time, toxicity and increasing morbidity are factors that limit the use of these drugs. OBJECTIVES: To describe the response to radiofrequency thermotherapy in the treatment of localized cutaneous leishmaniasis in Brazil, and to evaluate its safety and tolerability. METHODS: We conducted a non-comparative open trial with a total of 15 patients confirmed to have cutaneous leishmaniasis on parasitological examination. A single radiofrequency thermotherapy session at 50ºC for 30 seconds was applied to the lesion and its edges. In patients with more than one lesion, only the largest one was treated initially. If after 30 days there was no evidence of healing, the smaller lesion was also treated with thermotherapy. Clinical cure was defined as visible healing for three months after treatment. The patients were followed-up for six months and there was no follow-up loss. RESULTS: Of all 23 lesions, only two evolved to complete healing without the need of treatment. Of 21 lesions, 18 (85.7%) achieved full healing. The main observed side effects were itching, burning sensation, pain and blisters. STUDY LIMITATIONS: Sample with a small number of patients and short follow-up. CONCLUSION: Thermotherapy can be considered a therapeutic alternative in localized cutaneous leishmaniasis, especially in cases of single cutaneous lesions and with formal contraindications to conventional treatment with pentavalent antimonials.

Efficacy of pentavalent antimoniate intralesional infiltration therapy for cutaneous leishmaniasis: A systematic review.

BACKGROUND: The mainstays of cutaneous leishmaniasis (CL) treatment, in several world regions, are pentavalent antimony (Sbv) compounds administered parenterally, despite their recognized toxicity, which requires frequent laboratory monitoring and complicates their use in areas with scarce infrastructure. As result of these drawbacks, the WHO Expert Committee on leishmaniasis has expanded the recommendations for the use of local therapies, including Sbv intralesional infiltration (IL-Sbv), as CL therapy alternatives even in the New World. However, the efficacy of these approaches has never been compiled. The aim of this study was to critically and systematically assess the efficacy of IL-Sbv for CL treatment. METHODOLOGY: The PRISMA guidelines for systematic reviews and the Cochrane manual were followed. The sources used were the MEDLINE and LILACS databases and the International Clinical Trials Registry Platform of the World Health Organization. The outcome of interest was a clinical cure, defined as complete re-epithelialization of all lesions. The IL-Sbv pooled cure rate was estimated for several subgroups and direct comparisons were performed when possible. RESULTS: Thirty nine articles (40 studies) involving 5679 patients treated with IL-Sbv infiltration were included. In direct comparison, only three studies involving 229 patients compared IL-Sbv infiltration versus placebo and no difference was observed (OR: 1,9; 95%IC 0,93 to 3,82) based on cure rate 69.6% (95%CI 17.6-96.1%) and 83,2% (95%CI 66-92.7%) for placebo and IL-Sbv, respectively. In an alternative and non-comparative analysis, gathering all study arms using the intervention, the pooled IL-Sbv efficacy rate was 75% (95%CI 68-81%). In the Old World, the observed overall IL-Sbv efficacy rate was 75% (95%CI 66-82%), and the cure rates were significantly higher with sodium stibogluconate (SSG) than with meglumine antimoniate (MA): 83% (95%CI 75-90%) versus 68% (95%CI 54-79%), p = 0.03. Studies directly comparing IL-Sbv with topical 15% paromomycin ointment, IL hypertonic saline, radiofrequency-induced heat therapy, topical trichloroacetic acid and cryotherapy showed no significant difference in efficacy between the interventions. The analyses suggested a higher efficacy of IL-Sbv combined with cryotherapy (81.8%, 95%IC 62.4-92.4%) when compared with IL-Sbv alone (53.3%, 95%IC 46.1-66%), OR: 3.14 (95%CI 1.1-8.9), p = 0.03. In the New World, the global IL-Sbv efficacy was 77%(95%CI 66-85%). In contrast with the Old World, a significant difference favoring MA in relation to SSG was observed: 61% (95%CI 49-73%) versus 82% (95%CI 70-89%).By comparing IL infiltration schedules, it was determined that patients submitted to IL-Sbv treatments longer than 14 days had higher cure rates. CONCLUSIONS: Despite the high heterogeneity and low methodological quality of studies, an indirect comparison shows that the antimony infiltration efficacy rate is similar to that reported for antimony systemic use. The evidence gathered thus far is insufficient to identify the ideal IL therapeutic regime or estimate the rates of adverse events and mucosal late complications.

Efficacy, Safety and Cost-Effectiveness of Thermotherapy in the Treatment of Leishmania donovani-Induced Cutaneous Leishmaniasis: A Randomized Controlled Clinical Trial.

Leishmania donovani causes cutaneous leishmaniasis (CL) in Sri Lanka. Standard treatment is multiple, painful doses of intralesional sodium stibogluconate (IL-SSG). Treatment failures are increasingly reported, hence the need to investigate alternatives. Efficacy, safety, and cost-effectiveness of thermotherapy were assessed for the first time for L. donovani CL. A single blinded noninferiority randomized controlled trial was conducted on new laboratory-confirmed CL patients with single lesions (N = 213). Selected patients were randomly assigned to 1) test group (N = 98; single session of radiofrequency-induced heat therapy (RFHT) given at 50°C for 30 seconds) and 2) control group (N = 115; 1-3 mL IL-SSG given weekly, until cure/10 doses). Patients were followed-up fortnightly for 12 weeks to assess clinical cure. Cost of treatment was assessed using scenario building technique. Cure rates by 8, 10, and 12 weeks in RFHT group were 46.5%, 56.5%, and 65.9% as opposed to 28%, 40.8%, and 59.4% in IL-SSG group, with no major adverse events. Cure rate by RFHT was significantly higher at 8 weeks (P = 0.009, odds ratio [OR]: 2.236, confidence interval [CI]: 1.217-4.108) and 10 weeks (P = 0.035, OR: 1.881, CI: 1.044-3.388), but comparable thereafter. Cost of RFHT was 7 times less (USD = 1.54/patient) than IL-SSG (USD = 11.09/patient). A single application of RFHT is safe, cost-effective, and convenient, compared with multiple doses of IL-SSG in the treatment of L. donovani CL. Therefore, RFHT would be considered noninferior as per trial outcome when compared with standard IL-SSG therapy with multiple benefits for the patient and the national health care system.

M1 homeopathic complex trigger effective responses against Leishmania (L) amazonensis in vivo and in vitro.

Leishmaniasis is a term referring to a range of clinical conditions caused by protozoan parasites of the genus Leishmania, Trypanosomatidae family, Kinetoplastida order that is transmitted by the bite of certain species of mosquitoes Phlebotominae subfamily. These parasites infect hosts wild and domestic mammals, considered as natural reservoirs and can also infect humans. Leishmania are obligate intramacrophage protozoa that have exclusively intracellular life style. This suggests that the amastigotes possess mechanisms to avoid killing by host cells. Cutaneous leishmaniasis, the most common form of the disease, causes ulcers on exposed parts of the body, leading to disfigurement, permanent scars, and stigma and in some cases disability. Many studies concluded that the cytokines profile and immune system of host have fundamental role in humans and animals natural self-healing. Conventional treatments are far from ideals and the search for new therapeutic alternatives is considered a strategic priority line of research by the World Health Organization. A promising approach in the field of basic research in homeopathy is the treatment of experimental infections with homeopathic drugs prepared from natural substances associations highly diluted, which comprise a combination of several different compounds considered as useful for a symptom or disease. Therefore, this study aimed to evaluate the effect of M1, a complex homeopathic product, in macrophage-Leishmania interaction in vitro and in vivo. It was used RAW cells lineage and BALB/c mice as a host for the promastigotes of L. amazonensis (WHOM/BR/75/Josefa). Several biochemical and morphological parameters were determined. Together, the harmonic results obtained in this study indicate that, in general, the highly diluted products trigger rapid and effective responses by living organisms, cells and mice, against Leishmania, by altering cytokines profile, by NO increasing (p<0.05), by decreasing parasitic load (p<0.001), and modifying classical maturation and biogenesis of parasitophorous vacuoles (p<0.001). M1 complex decreased endocytic index (p<0.001), and the % of infected macrophages (p<0.05), preventing the development of lesions (p<0.05) caused by L. amazonensis by increasing Th1 response (p<0.05). Therefore the M1complex can be a good candidate for a complementary therapy to conventional treatments, since all the parameters observed in vitro and in vivo improved. It could be an interesting clinical tool in association to a classical anti-parasitic treatment, maybe resulting in better quality of life to the patients, with less toxicity.

Clinical features and treatment response of cutaneous leishmaniasis in North-West Ethiopia.

OBJECTIVE: Cutaneous leishmaniasis (CL) receives far less attention than visceral leishmaniasis. Nevertheless, CL is predominantly caused by a unique species in Ethiopia (L. aethiopica), which is known to cause severe forms such as diffuse (DCL) and mucocutaneous leishmaniasis (MCL). We report on the number and type of CL cases diagnosed, the clinical features, the treatments and treatment outcomes in North-West Ethiopia. METHODS: This is a retrospective chart record analysis of CL patients treated at the Leishmania Research and Treatment Center (LRTC) of the University of Gondar, Ethiopia. RESULTS: From 178 CL patients seen between January 2014 and December 2015, a total of 154 chart records were retrieved. These included 80 localised CL (LCL), 7 DCL and 67 MCL. The median age was 23 years; 71.4% were male. Most (n = 121, 78.6%) of the lesions were on the face. The median time since onset was 12 months (6-24 months), and 28.6% presented after a trial of traditional medicine. The treatment of all forms of CL mainly consisted of 30 days of IM antimonial injections. Of these, 51/133 (38.3%) required treatment extension or change due to nonresponse. Three cases were treated with liposomal amphotericin B or miltefosine (two received the combination), of which two responded well. CONCLUSION: CL was found to be complicated and difficult to treat. MCL was common, and patients presented after long delays. There is an urgent need to look for better treatment options for CL and improve access to care.

Multi-Anti-Parasitic Activity of Arylidene Ketones and Thiazolidene Hydrazines against Trypanosoma cruzi and Leishmania spp.

A series of fifty arylideneketones and thiazolidenehydrazines was evaluated against Leishmania infantum and Leishmania braziliensis. Furthermore, new simplified thiazolidenehydrazine derivatives were evaluated against Trypanosoma cruzi. The cytotoxicity of the active compounds on non-infected fibroblasts or macrophages was established in vitro to evaluate the selectivity of their anti-parasitic effects. Seven thiazolidenehydrazine derivatives and ten arylideneketones had good activity against the three parasites. The IC50 values for T. cruzi and Leishmania spp. ranged from 90 nM-25 µM. Eight compounds had multi-trypanocidal activity against T. cruzi and Leishmania spp. (the etiological agents of cutaneous and visceral forms). The selectivity of these active compounds was better than the three reference drugs: benznidazole, glucantime and miltefosine. They also had low toxicity when tested in vivo on zebrafish. Trying to understand the mechanism of action of these compounds, two possible molecular targets were investigated: triosephosphate isomerase and cruzipain. We also used a molecular stripping approach to elucidate the minimal structural requirements for their anti-T. cruzi activity.

Use of Optical Imaging Technology in the Validation of a New, Rapid, Cost-Effective Drug Screen as Part of a Tiered In Vivo Screening Paradigm for Development of Drugs To Treat Cutaneous Leishmaniasis.

In any drug discovery and development effort, a reduction in the time of the lead optimization cycle is critical to decrease the time to license and reduce costs. In addition, ethical guidelines call for the more ethical use of animals to minimize the number of animals used and decrease their suffering. Therefore, any effort to develop drugs to treat cutaneous leishmaniasis requires multiple tiers of in vivo testing that start with higher-throughput efficacy assessments and progress to lower-throughput models with the most clinical relevance. Here, we describe the validation of a high-throughput, first-tier, noninvasive model of lesion suppression that uses an in vivo optical imaging technology for the initial screening of compounds. A strong correlation between luciferase activity and the parasite load at up to 18 days postinfection was found. This correlation allows the direct assessment of the effects of drug treatment on parasite burden. We demonstrate that there is a strong correlation between drug efficacy measured on day 18 postinfection and the suppression of lesion size by day 60 postinfection, which allows us to reach an accurate conclusion on drug efficacy in only 18 days. Compounds demonstrating a significant reduction in the bioluminescence signal compared to that in control animals can be tested in lower-throughput, more definitive tests of lesion cure in BALB/c mice and Golden Syrian hamsters (GSH) using Old World and New World parasites.

Efficacy of fractional CO2 laser in treatment of atrophic scar of cutaneous leishmaniasis.

Cutaneous leishmaniasis is an endemic disease in Iran. Unfortunately, it can lead to unsightly atrophic scars with limited treatment options. Fractional CO2 laser is accepted for treatment of atrophic acne scars and recently has been used to treat cutaneous leishmaniasis, so we planned to use fractional CO2 laser on leishmaniasis scar. We conducted this study on 60 leishmaniasis scars on the face of 40 patients. The lesions were treated by a fractional CO2 laser with beam size of 120 µm, with energy of 50-90 mJ, and 50-100 spots/cm(2) density with two passes in three monthly sessions. Evaluation was done in the first and second months after the first treatment and 3 and 6 months after the last treatment. Digital photography was performed at each visit. Assessment of improvement rate by patient and physician was rated separately as follows: no improvement (0%), mild (<25%), moderate (25-50%), good (51-75%), and excellent (76-100%). Based on patients' opinion, in the first and second follow-up, 48.3 and 90% of them reported moderate to excellent healing, respectively (p < 0.001). In 3 and 6 months follow-up after the end of the experiment, most of the patients (88.3 and 95%, respectively) reported moderate to excellent healing of scars. Based on two observers' opinion, healing in the first follow-up in most of the patients (65%) was mild to moderate and 33% were reported as having no healing. In the second follow-up, only 5% of the patients were reported with no healing and 60% were reported as having moderate healing (p < 0.001). In 3 and 6 months follow-up, most of the patients (95 and 96.6%) were reported as having moderate to excellent healing (p = <0.001). Our results underlined the high efficacy of fractional CO2 laser for leishmaniasis scar. No significant adverse effects were noted.

The Effect of Ursolic Acid on Leishmania (Leishmania) amazonensis Is Related to Programed Cell Death and Presents Therapeutic Potential in Experimental Cutaneous Leishmaniasis.

Among neglected tropical diseases, leishmaniasis is one of the most important ones, affecting more than 12 million people worldwide. The available treatments are not well tolerated, and present diverse side effects, justifying the search for new therapeutic compounds. In the present study, the activity of ursolic acid (UA) and oleanolic acid (OA) were assayed in experimental cutaneous leishmaniasis (in vitro and in vivo). Promastigote forms of L. amazonensis were incubated with OA and UA for 24h, and effective concentration 50% (EC50) was estimated. Ultraestructural alterations in Leishmania amazonensis promastigotes after UA treatment were evaluated by transmission electron microscopy, and the possible mode of action was assayed through Annexin V and propidium iodide staining, caspase 3/7 activity, DNA fragmentation and transmembrane mitochondrial potential. The UA potential was evaluated in intracellular amastigotes, and its therapeutic potential was evaluated in L. amazonensis infected BALB/c mice. UA eliminated L. amazonensis promastigotes with an EC50 of 6.4 µg/mL, comparable with miltefosine, while OA presented only a marginal effect on promastigote forms at 100 µg/mL. The possible mechanism by which promastigotes were eliminated by UA was programmed cell death, independent of caspase 3/7, but it was highly dependent on mitochondria activity. UA was not toxic for peritoneal macrophages from BALB/c mice, and it was able to eliminate intracellular amastigotes, associated with nitric oxide (NO) production. OA did not eliminate amastigotes nor trigger NO. L. amazonensis infected BALB/c mice submitted to UA treatment presented lesser lesion size and parasitism compared to control. This study showed, for the first time, that UA eliminate promastigote forms through a mechanism associated with programed cell death, and importantly, was effective in vivo. Therefore, UA can be considered an interesting candidate for future tests as a prototype drug for the treatment of cutaneous leishmaniasis.

Lymphatic Dissemination in Cutaneous Leishmaniasis Following Local Treatment.

Cutaneous leishmaniasis (CL) is diverse in its clinical presentation but usually demonstrates an erythematous, infiltrated, ulcerated, and crusted papule or nodule in exposed areas of the body. Rare clinical features have been reported including lymphatic dissemination, usually with subcutaneous nodules along lymphatic channels. Herein, we present six patients suffering from Old World CL with lymphatic dissemination characterized by sporotrichoid subcutaneous nodules along the lymphatic channels draining the primary lesion. Patients' history, clinical and laboratory findings were collected and summarized. Lymphatic dissemination of CL in our patients manifested as subcutaneous nodules without epidermal involvement within the axis of lymphatic drainage toward the regional lymph node, at times accompanied by regional lymphadenopathy. In all patients, the lymphatic dissemination was not present at initial diagnosis of CL, appearing only after local (topical or intralesional) treatment was initiated. In three patients, the subcutaneous nodules resolved without systemic treatment. Lymphatic dissemination of Old World CL is not uncommon and may possibly be triggered by local treatment. It should be recognized by dermatologists, especially those working in endemic areas. Systemic treatment may be not necessary since spontaneous resolution may occur.

Assessment of ß-lapachone loaded in lecithin-chitosan nanoparticles for the topical treatment of cutaneous leishmaniasis in L. major infected BALB/c mice.

Patients affected by cutaneous leishmaniasis need a topical treatment which cures lesions without leaving scars. Lesions are produced not only by the parasite but also by an uncontrolled and persistent inflammatory immune response. In this study, we proposed the loading of ß-lapachone (ß-LP) in lecithin-chitosan nanoparticles (NP) for targeting the drug to the dermis, where infected macrophages reside, and promote wound healing. Although the loading of ß-LP in NP did not influence the drug antileishmanial activity it was critical to achieve important drug accumulation in the dermis and permeation through the skin. When topically applied in Leishmania major infected BALB/c mice, ß-LP NP achieved no parasite reduction but they stopped the lesion progression. Immuno-histopathological assays in CL lesions and quantitative mRNA studies in draining lymph nodes confirmed that ß-LP exhibited anti-inflammatory activity leading to the down-regulation of IL-1ß and COX-2 expression and a decrease of neutrophils infiltrate. FROM THE CLINICAL EDITOR: Cutaneous leishmaniasis often leaves patients with unsightly scars due to the body's inflammatory response to the infection. The authors in this paper described topical treatment using ß-lapachone (ß- LP) loaded in lecithin-chitosan nanoparticles (NP) in an animal model. Results confirmed the reduction of inflammatory response without affecting the parasite killing efficacy. These findings would pave way for further clinical testing in the near future.

T cell lipid peroxidation induces ferroptosis and prevents immunity to infection.

The selenoenzyme glutathione peroxidase 4 (Gpx4) is a major scavenger of phospholipid hydroperoxides. Although Gpx4 represents a key component of the reactive oxygen species-scavenging network, its relevance in the immune system is yet to be defined. Here, we investigated the importance of Gpx4 for physiological T cell responses by using T cell-specific Gpx4-deficient mice. Our results revealed that, despite normal thymic T cell development, CD8(+) T cells from T(ΔGpx4/ΔGpx4) mice had an intrinsic defect in maintaining homeostatic balance in the periphery. Moreover, both antigen-specific CD8(+) and CD4(+) T cells lacking Gpx4 failed to expand and to protect from acute lymphocytic choriomeningitis virus and Leishmania major parasite infections, which were rescued with diet supplementation of high dosage of vitamin E. Notably, depletion of the Gpx4 gene in the memory phase of viral infection did not affect T cell recall responses upon secondary infection. Ex vivo, Gpx4-deficient T cells rapidly accumulated membrane lipid peroxides and concomitantly underwent cell death driven by ferroptosis but not necroptosis. These studies unveil an essential role of Gpx4 for T cell immunity.

Liposomal-lupane system as alternative chemotherapy against cutaneous leishmaniasis: macrophage as target cell.

Leishmania amazonensis causes human diseases that range from self-healing to diffusion cutaneous lesions. The chemotherapy of leishmaniasis requires long-term treatment and has been based on the use of pentavalent antimonials. Liposomes have been used as antileishmanial drug carries and have adjuvant activity in vaccines against several microorganisms, representing an important option to the development of new therapeutics for the disease. In this study, we developed a liposomal formulation containing lupane [3ß,6ß,16ß-trihydroxylup-20(29)-ene], isolated from fruits of Combretum leprosum with pharmacological properties as antinociceptive, anti-inflammatory, antiulcerogenic and antileishmanial activities. The aim of the present study was to evaluate the efficacy of liposomal-lupane in L. amazonensis-infection model. Liposomes were prepared by the extrusion method with DPPC, DPPS and cholesterol at 5:1:4 weight ratio. The lupane (2 mg/mL) was added to the lipid mixture, solubilized in chloroform and dried under nitrogen flow. The activity of liposomal-lupane was conducted in vitro with mouse peritoneal infected macrophages. Furthermore, mice were infected in the right hind footpad with 10(5) stationary growth phase of L. amazonensis promastigotes. After 6 weeks, animals were treated with liposomal-lupane for 15 days by intraperitoneal injection. The evolution of disease was monitored weekly by measuring footpad thickness with a caliper. Three days after the treatment, peritoneal macrophages were collected, plated and production of the cytokines IL-10 and IL-12 was evaluated in supernatants of the cultures after 24 h. The results indicate that the liposomal system containing lupane achieved here is a promising tool to confer antileishmanial activity to infected macrophages.

Influence of the nutritional status in the clinical and therapeutical evolution in adults and elderly with American Tegumentary Leishmaniasis.

The objective of this study is to describe the nutritional status of adult and elderly patients with American Tegumentary Leishmaniasis (ATL). It was conducted a longitudinal study in 68 adult and elderly patients with ATL treating at the Surveillance Leishmaniasis Laboratory at the Evandro Chagas Clinical Research Institute, Oswaldo Cruz Foundation (Fiocruz), from 2009 to 2012. The nutritional assessment included the body mass index (BMI) and serum albumin levels. The clinical evolution (epithelialization and wound healing) was measured up to two years after ATL treatment. Most of the sample was composed of men (71%), adults (73%), with household income of 1-5 minimum wages (79%), and incomplete elementary school (48.5%). The predominant ATL form was cutaneous (72%), and 39% presented comorbidities, the most frequent was hypertension (30.8%). The most prevalent clinical and nutritional events were: recent decrease in food intake (23.9%); nasal obstruction (22.1%); oral ulcer (14.7%), anorexia and dysphagia (13.2% each) and odynophagia (10.3%). The total healing time was 115.00 (IR=80-230) days for skin lesions, and 120.00 (IR=104.50-223.50) days for mucous membrane lesions. Low body weight in 10%, and hypoalbuminemia in 12% of the patients have been observed. Low body weight was associated with age, mucosal leishmaniasis (ML), nasal obstruction, recent decrease in food intake and hypoalbuminemia. As for serum albumin depletion, association with the ML, dyspnea, dysphagia, odynophagia, recent decrease in food intake, absence of complete healing of the skin lesions, and increased healing time for mucous membrane lesions, was observed. The ML and their events that affect the alimentary intake have been related to the impairment of the nutritional status. Additionally, serum albumin depletion negatively affected the healing of the lesions, suggesting that a nutritional intervention can increase the effectiveness of the ATL treatment.