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Visceral leishmaniasis (VL), caused by , is associated with parasite-induced immunological and physiological changes that ensure the survival of amastigotes within the host. Both the parasite and the host have nutritional requirements, and for auxotrophic , dependence on the host to supply specific growth requirements is essential. This highlights an intricate link between host immunity and metabolism during VL. This study explores the interplay between the host metabolome and immune responses pre- and post-infection and treatment, aiming to identify early metabolite markers of therapeutic success against . BALB/c mice infected with were divided into cured and non-cured groups based on treatment with a non-ionic surfactant vesicle formulation of sodium stibogluconate (300 mg Sb /kg, SSG-NIV) or PBS vehicle control. Specific immune responses were determined at day 21 and day 60 post-infection, and serum metabolite levels was measured using untargeted GC×GC-TOFMS metabolomics. Treatment effectively reduced parasite loads, triggering heightened CD4+ and CD8+ T-cell responses at day 21, with increased IFN-γ, IL-12, and IL-4, and decreased IL-10 and TGF-β. Pre-treatment metabolomics analysis identified changes in glycolysis, fatty acid and amino acid metabolism 1-week PI, suggesting an increased Warburg effect to supplement parasite survival and initiation of immune responses. Valine, lactic acid, and glycerol-1-oleate were identified as markers of early infection. Treatment with SSG-NIV altered metabolism of major macromolecules and the TCA cycle relative to non-cured groups. Additionally, glycine and ribitol show promise as immune correlates for antiparasitic therapies. These findings highlight the diagnostic and prognostic potential of serum-derived metabolites in monitoring host immune responses to VL and treatment.

Leishmaniasis is a disease caused by the protozoan Leishmania that resides mainly in mononuclear phagocytic system tissues. Pentavalent antimonials are the main treatment option, although these drugs have toxic side effects and high resistance rates. A potentially alternative and more effective therapeutic strategy is to use liposomes as carriers of the antileishmanial agents. The aims of this study were to develop antimonial drugs entrapped into phosphatidylserine liposomes and to analyze their biological and physicochemical characteristics. Liposomes containing meglumine antimoniate (MA) or pentavalent antimony salt (Sb) were obtained through filter extrusion (FEL) and characterized by transmission electron microscopy. Promastigotes of Leishmania infantum were incubated with the drugs and the viability was determined with a tetrazolium dye (MTT assay). The effects of these drugs against intracellular amastigotes were also evaluated by optical microscopy, and mammalian cytotoxicity was determined by an MTT assay. Liposomes had an average diameter of 162nm. MA-FEL showed inhibitory activity against intracellular L. infantum amastigotes, with a 50% inhibitory concentration (IC50) of 0.9μg/mL, whereas that of MA was 60μg/mL. Sb-FEL showed an IC50 value of 0.2μg/mL, whereas that of free Sb was 9μg/mL. MA-FEL and Sb-FEL had strong in vitro activity that was 63-fold and 39-fold more effective than their respective free drugs. MA-FEL tested at a ten-times higher concentration than Sb-FEL did not show cytotoxicity to mammalian cells, resulting in a higher selectivity index. Antimonial drug-containing liposomes are more effective against Leishmania-infected macrophages than the non-liposomal drugs.

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. 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. 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. 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.

Aerobic capacity plays a crucial role in football performance, making it a focal point in training processes. Small-sided games (SSGs) are widely used in football training, but the relationship between aerobic capacity and running performance during SSGs remains unclear. The aim of this study was to investigate possible correlations between maximum oxygen uptake (VO2max) and running performance in youth football players in SSGs (4:4, 3:3, 2:2, 1:1) with three different pitch sizes per player (150, 100, 75 m2/player). Sixteen male U15 football players participated in the study. Players underwent the Yo-Yo intermittent recovery test level 1, and their VO2max was estimated based on their performance. Subsequently, players participated in SSGs wearing GPS devices to measure internal and external load. Pearson or Spearman correlation was applied for statistical analysis depending on the normal distribution of the data. The results reveal that, for 4:4 and 3:3 relationships, larger pitches led to a greater impact of aerobic capacity (total distance (TD): 4:4, 150 m2/pl, r = 0.715, p = 0.002; 100 m2/pl, r = 0.656, p = 0.006; 75 m2/pl, r = 0.586, p = 0.017). In the 2:2 relationship, the opposite was observed, with more correlations appearing on smaller pitches (TD: 2:2, 100 m2/pl, r = 0.581, p = 0.018; 75 m2/pl, r = 0.747, p < 0.001). In the 1:1 relationship, correlations with VO2max, total distance, and speed were observed only on the larger pitch. In conclusion, the aerobic capacity of young football players can influence running performance indicators in SSGs. Therefore, aerobic capacity could serve as a criterion for team composition, making SSGs more competitive. Additionally, the variation in correlations in the 2:2 relationship and their limited presence in the 1:1 relationship may be attributed to technical–tactical factors, such as increased ball contacts and one-on-one situations typically occurring in smaller setups.

Background A previously published proof of principle phase IIa trial with 113 patients from Kabul showed that bipolar high-frequency (HF) electro-cauterization (EC) of cutaneous leishmaniasis (CL) ulcers and subsequent moist wound treatment (MWT) closed 85% of all Leishmania (L.) tropica lesions within 60 days. Methods A three-armed phase IIb, randomized and controlled clinical trial was performed in Mazar-e-Sharif. L. tropica - or L. major -infected CL patients received intradermal sodium stibogluconate (SSG) (Group I); HF-EC followed by MWT with 0.045% DAC N-055 (Group II); or MWT with 0.045% DAC N-055 in basic crème alone (Group III). The primary outcome was complete epithelialisation before day 75 after treatment start. Results 87 patients enrolled in the trial were randomized into group I (n = 24), II (n = 32) and III (n = 31). The per-protocol analysis of 69 (79%) patients revealed complete epithelialisation before day 75 in 15 (of 23; 65%) patients of Group I, in 23 (of 23; 100%) patients of Group II, and in 20 (of 23; 87%) patients of Group III (p = 0.004, Fisher’s Exact Test). In the per-protocol analysis, wound closure times were significantly different between all regimens in a pair-wise comparison (p = 0.000039, Log-Rank (Mantel-Cox) test). In the intention-to-treat analysis wound survival times in Group II were significantly different from those in Group I (p = 0.000040, Log-Rank (Mantel-Cox) test). Re-ulcerations occurred in four (17%), three (13%) and seven (30%) patients of Group I, II or III, respectively (p = 0.312, Pearson Chi-Square Test). Conclusions Treatment of CL ulcers with bipolar HF-EC followed by MWT with 0.045% DAC N-055 or with DAC N-055 alone showed shorter wound closure times than with the standard SSG therapy. The results merit further exploration in larger trials in the light of our current knowledge of in vitro and in vivo activities of chlorite. Clinicaltrials.gov ID: NCT00996463. Registered: 15 th October 2009.

Background In the last decade, resistance to antimonials has become a serious problem due to the emergence of drug-resistant strains. Therefore, understanding the mechanisms used by Leishmania parasites to survive under drug pressure is essential, particularly for species of medical-veterinary importance such as L. amazonensis . Methods Here, we used RNA-seq technology to analyse transcriptome profiles and identify global changes in gene expression between antimony-resistant and -sensitive L. amazonensis promastigotes. Results A total of 723 differentially expressed genes were identified between resistant and sensitive lines. Comparative transcriptomic analysis revealed that genes encoding proteins involved in metabolism (fatty acids) and stress response, as well as those associated with antimony resistance in other Leishmania species, were upregulated in the antimony-resistant line. Most importantly, we observed upregulation of genes encoding autophagy proteins, suggesting that in the presence of trivalent stibogluconate (Sb III ) L. amazonensis can activate these genes either as a survival strategy or to induce cell death, as has been observed in other parasites. Conclusions This work identified global transcriptomic changes in an in vitro -adapted strain in response to Sb III . Our results provide relevant information to continue understanding the mechanism used by parasites of the subgenus Leishmania ( L. amazonensis ) to generate an antimony-resistant phenotype.

Drugs based on pentavalent antimony are first-line treatment of the parasite disease leishmaniasis. It is generally believed that Sb(V) acts as a prodrug, which is activated by reduction to Sb(III); however, the site of reduction is not known. It has been hypothesised that the reduction takes place in the parasites’ host cells, the macrophages. In this study, the human macrophage cell line Mono Mac 6 was exposed to Sb(V) in form of the drug sodium stibogluconate (Pentostam™). Cell extracts were analysed for Sb species by high-performance liquid chromatography with inductively coupled plasma-mass spectrometry detection. We found that Sb(V) is actually reduced to Sb(III) in the macrophages; up to 23% of the intracellular Sb was found as Sb(III). Transfer of the cells to Sb-free medium rapidly decreased their Sb(V) and Sb(III) content. Induction of the cell’s production of reactive oxygen species did not have any marked effect on the intracellular amounts of Sb(III).

Leishmania is a digenetic protozoan parasite causing leishmaniasis in humans. The different clinical forms of leishmaniasis are caused by more than twenty species of Leishmania that are transmitted by nearly thirty species of phlebotomine sand flies. Pentavalent antimonials (such as Pentostam or Glucantime) are the first line drugs for treating leishmaniasis. Recent studies suggest that pentavalent antimony (Sb(V)) acts as a pro-drug, which is converted to the more active trivalent form (Sb(III)). However, sensitivity to trivalent antimony varies among different Leishmania species. In general, Leishmania species causing cutaneous leishmaniasis (CL) are more sensitive to Sb(III) than the species responsible for visceral leishmaniasis (VL). Leishmania aquaglyceroporin (AQP1) facilitates the adventitious passage of antimonite down a concentration gradient. In this study, we show that Leishmania species causing CL accumulate more antimonite, and therefore exhibit higher sensitivity to antimonials, than the species responsible for VL. This species-specific differential sensitivity to antimonite is directly proportional to the expression levels of AQP1 mRNA. We show that the stability of AQP1 mRNA in different Leishmania species is regulated by their respective 3'-untranslated regions. The differential regulation of AQP1 mRNA explains the distinct antimonial sensitivity of each species.

Resistance of Leishmania donovani to pentavalent antimonials, the first-line treatment of visceral leishmaniasis (VL), has become a critical issue worldwide. Second-line and new drugs are also not devoid of limitations. Suitable drug-delivery systems can improve the mode of administration and action of the existing antimonials, thus increasing their clinical life. We investigated the efficacy of sodium stibogluconate (SSG) in phosphatidylcholine (PC)-stearylamine-bearing liposomes (PC-SA-SSG), PC-cholesterol liposomes (PC-Chol-SSG) and free amphotericin B (AmB) against SSG-resistant L. donovani strains in 8-wk infected BALB/c mice. Animals were sacrificed and parasites in liver, spleen and bone marrow were estimated 4-wk post-treatment by microscopic examination of stamp smears and limiting dilution assay. A set of PC-SA-SSG and AmB treated mice were further studied for protection against reinfection. Serum antibodies and cytokine profiles of ex-vivo cultured splenocytes were determined by ELISA. Uptake of free and liposomal SSG in intracellular amastigotes was determined by atomic absorption spectroscopy. Rhodamine 123 and 5-carboxyfluorescein, known substrates of Pgp and MRP transporter proteins, respectively, were used in free and liposomal forms for efflux studies to estimate intracellular drug retention. Unlike free and PC-Chol-SSG, PC-SA-SSG was effective in curing mice infected with two differentially originated SSG-unresponsive parasite strains at significantly higher levels than AmB. Successful therapy correlated with complete suppression of disease-promoting IL-10 and TGF-β, upregulation of Th1 cytokines and expression of macrophage microbicidal NO. Cure due to elevated accumulation of SSG in intracellular parasites, irrespective of SSG-resistance, occurs as a result of increased drug retention and improved therapy when administered as PC-SA-SSG versus free SSG. The design of this single-dose combination therapy with PC-SA-SSG for VL, having reduced toxicity and long-term efficacy, irrespective of SSG-sensitivity may prove promising, not only to overcome SSG-resistance in Leishmania, but also for drugs with similar resistance-related problems in other diseases.

We evaluated the leishmanicidal activity of commercially available 5α-cholest-7-en-3β-ol [5α-chol], (+)-4-cholesten-3-one [(+)-4-chol] and the equimolar mixture of the two of them in promastigotes and amastigotes of two different strains of Leishmania mexicana (LCL) and (DCL). The leishmanicidal effectiveness of these sterols was determined by promastigote growth-kinetic experiments and promastigote viability using the propidium iodide staining procedure. The proliferation test was performed using the CFSE (5-Carboxyfluorescein N-succinimidyl ester) staining of parasites at different time points. To determine the leishmanicidal effectiveness of these sterols in amastigotes, we evaluated parasite killing inside of macrophages at different time points. The trypan blue exclusion test was used to determine cytotoxicity of sterols in uninfected macrophages. We included in all experiments a control group of parasites treated with 2% DMSO (Dimethyl Sulfoxide) and another one treated with the reference drug sodium stibogluconate (Sb). Our results showed that the equimolar mixture at 2000 times lower concentration presented similar leishmanicidal activity as Sb. This mixture was similarly effective at 100 times lower concentration than individual sterols tested separately indicating the existence of a synergistic effect against LCL and DCL parasites. The therapeutic index of the equimolar mixture was 10,000—16,000 times higher than the one recorded by Sb and was not cytotoxic to macrophages. Therefore, the equimolar mixture of 5α-Chol and (+)-4-chol may represent a potential alternative for the treatment of cutaneous leishmaniasis.