Topical application of ozonated sunflower oil accelerates the healing of lesions of cutaneous leishmaniasis in mice under meglumine antimoniate treatment.

Besides being scarce, the drugs available for treating cutaneous leishmaniasis have many adverse effects. Ozone is an option to enhance the standard treatment due to the wound-healing activity reported in the literature. In this study, we evaluated the efficiency of ozonated sunflower oil as an adjuvant in treating cutaneous lesions caused by Leishmania amazonensis. BALB/c mice were infected with L. amazonensis, and after the lesions appeared, they were treated in four different schedules using the drug treatment with meglumine antimoniate (Glucantime®), with or without ozonated oil. After thirty days of treatment, the lesions' thickness and their parasitic burden, blood leukocytes, production of NO and cytokines from peritoneal macrophages and lymph node cells were analyzed. The group treated with ozonated oil plus meglumine antimoniate showed the best performance, improving the lesion significantly. The parasitic burden showed that ozonated oil enhanced the leishmanicidal activity of the treatment, eliminating the parasites in the lesion. Besides, a decrease in the TNF levels from peritoneal macrophages and blood leukocytes demonstrated an immunomodulatory action of ozone in the ozonated oil-treated animals compared to the untreated group. Thus, ozonated sunflower oil therapy has been shown as an adjuvant in treating Leishmania lesions since this treatment enhanced the leishmanicidal and wound healing effects of meglumine antimoniate.

Antiprotozoal potential of Vismia species (Hypericaceae), medicinal plants used to fight cutaneous leishmaniasis.

ETHNOPHARMACOLOGICAL RELEVANCE: Species of Vismia (Hypericaceae), known in Brazil as "lacre", are commonly used in traditional Amazonian medicine for the treatment of skin lesions, including those caused by Leishmania infection. AIM OF THE STUDY: Hexane extracts from the leaves of Vismia cayennensis, V. gracilis, V. sandwithii and V. guianensis, as well as from the fruits of the latter, in addition to the anthraquinones vismiaquinone, physcion and chrysophanol isolated from these species were explored for their anti-promastigote and anti-amastigote activity on Leishmania amazonensis. MATERIALS AND METHODS: Extracts were prepared by static maceration with n-hexane. The compounds, isolated by chromatographic techniques, were identified by spectroscopic methods (1H and 13C NMR). Promastigotes of L.amazonensis were incubated with hexane extracts (1-50 µg/mL) or anthraquinones (1-50 µM) and the parasite survival analyzed. The action of compounds on reactive oxygen species (ROS) production, mitochondrial membrane potential, and membrane integrity of promastigotes were evaluated by flow cytometer, and the cytotoxicity on mammalian cells using MTT assay. Furthermore, the activity of compounds against amastigotes and nitric oxide production were also investigated. RESULTS: Vismiaquinone and physcion were obtained from the leaves of V. guianensis. Physcion, as well as chrysophanol, were isolated from V. sandwithii. Vismia cayennensis and V. gracilis also showed vismiaquinone, compound detected in lower quantity in the fruits of V. guianensis. All extracts were active against the parasite, corroborating the popular use. The greatest activity against promastigotes was achieved with V. guianensis extract (IC50 4.3 µg/mL), precisely the most used Vismia species for treating cutaneous leishmaniasis. Vismiaquinone and physcion exhibited relevant activity with IC50 12.6 and 2.6 µM, respectively. Moreover, all extracts and anthraquinones tested induced ROS production, mitochondrial dysfunction, membrane disruption and were able to kill intracellular amastigote forms, being worthy of further in vivo studies as potential antileishmanial drugs. CONCLUSIONS: The overall data achieved in the current investigation scientifically validate the traditional use of Vismia species, mainly V. guianensis, as an anti-Leishmania agent. Furthermore, the promising results presented here indicate species of Vismia as potentially useful resources of Brazilian flora for the discovery of therapeutic solutions for neglected diseases.

Photodynamic therapy of cationic and anionic BSA-curcumin nanoparticles on amastigotes of Leishmania braziliensis and Leishmania major and Leishmania amazonensis.

Cutaneous leishmaniasis is a neglected disease prevalent in tropical countries, and conventional treatment can cause several serious side effects. Photodynamic therapy (PDT) can be considered a promising treatment alternative, as it is non-invasive therapy that has no side effects and uses accessible and low-cost substances, such as curcumin. This study evaluated the PDT response with cationic and anionic BSA nanoparticles encapsulated with curcumin in macrophages infected with L. braziliensis, L. major, and L. amazonensis. The nanoparticle system was characterized using a steady-state technique, scanning electron microscopy (SEM) study, and its biological activity was evaluated using macrophage cell lines infected with different Leishmania species. All spectroscopy measurements demonstrated that BSA curcumin (BSACur) has good photophysical properties, and confocal microscopy shows that macrophages and protozoa internalized the nanoparticles. The viability test demonstrated that at low concentrations, such as 0.1, 0.7, and 1.0 µmol. L-1, there was a decrease in cell viability after PDT application. Furthermore, a decrease in the number of parasites recovered was observed in the PDT groups. The results allowed us to conclude that curcumin loaded into BSA nanoparticles may have potential application in drug delivery systems for PDT protocols, demonstrating reduced cell viability at lower concentrations than free curcumin.

Serum Zinc Level and Efficacy of Zinc Therapy in Cutaneous Leishmaniasis: a Systematic Review and Meta-analysis.

Cutaneous leishmaniasis is a parasitic skin disease prevalent in many parts of the world. Zinc has been investigated for its potential role in the immune response against Leishmania parasites. This study aimed to systematically review the literature and conduct meta-analyses to evaluate the serum zinc level and efficacy of zinc therapy in cutaneous leishmaniasis. A comprehensive search of electronic databases was performed to find studies reporting serum zinc levels and the efficacy of zinc therapy in cutaneous leishmaniasis. Meta-analyses were conducted using RevMan software (version 5.4), calculating the mean difference for serum zinc levels and risk ratio for the efficacy of zinc therapy. A total of 11 studies with 1009 participants were evaluated. Five of these studies, comprising 637 participants, examined serum zinc levels; the remaining six, involving 372 individuals, examined the effectiveness of zinc therapy in treating cutaneous leishmaniasis. The results showed that the serum zinc level was significantly lower in cutaneous leishmaniasis patients compared to controls (MD: - 26.65; 95% CI: [- 42.74, - 10.57]; p = 0.001). However, zinc therapy did not demonstrate a significant clinical improvement compared to standard treatment (RR: 0.96; 95% CI: [0.74, 1.23], p = 0.73).

Antileishmaniasis effects of monoherbal skin ointment made from bark and leaf of Holoptelea integrifolia (Roxb.) Planch. in patients of cutaneous leishmaniasis.

Prevalence of parasitic skin diseases, cutaneous leishmaniasis is very frequent in Pakistan, especially in Baluchistan where occurrence of phelbotomine sand flies (vector of protozoan parasite of genus Leishmania) is very common. This study was conducted to assess the antileishmaniasis activity of Intericutol plus ointment (2% and 3%) made with ethanolic extract of bark and leaves of Holoptelea integrifolia (Roxb.)Planch. The antileishmaniasis effect was observed by applying the ointments on the lesions of leishmaniasis patients. 150 patients were included in the study that had single and multiple painful wet and dry ulcerative lesions on the exposed area of the body. The patients were divided into two groups of 75 patients each, applying 2% and 3% ointment respectively for 28 days. Dose of 10gm/week was applied topically on wounds. Excellent antileishmaniasis effect was observed with significant quick healing properties in the patients receiving 3% Intericutol plus ointment as compare to 2% showing 89% and 81% cure (p=0.02) respectively. It can be concluded that ointment made from medicinal plant proved to be very effective in treating cutaneous Leishmaniasis.

Antileishmanial effects, cellular mechanisms, and cytotoxicity of Elettaria cardamomum essential oil against Leishmania major infection.

Leishmaniasis is an infectious disease with various clinical manifestations. We studied the therapeutic effects of Elettaria cardamomum essential oil (ECEO) against Leishmania major infection. In vitro effects of ECEO against L. major were examined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and macrophage assays. Nitric oxide (NO) production, infection inhibition in macrophages, and the apoptotic activity of ECEO in treated parasites were also measured. By calculating the 50% cytotoxic concentrations (CC50), we studied the cytotoxicity effects of ECEO on human macrophage cells (THP-1). The efficacy of ECEO for improving cutaneous leishmaniasis (CL) lesions in mice (BALB/c) was determined by evaluating the size of lesions and the number of amastigotes before and after four weeks of treatment. The effects of ECEO on liver and kidney function in the tested mice were also evaluated. ECEO dose-dependently (p<0.001) inhibited the viability and the mean number of promastigotes and amastigote forms of L. tropica. Four weeks of treatment with ECEO at the doses of 2.5 and 5 mg/kg/ day significantly (p<0.001) improved the CL lesions and reduced the number of parasites in the infected mice. ECEO significantly increased NO production, apoptosis induction, and infection rate in parasites. The CC50 value for ECEO and MA was 303.4 µg/mL and 835.2 µg/mL, respectively. In the mice receiving ECEO at the doses of 2.5 and 5 mg/kg/day for 28 days, no significant change was reported between the serum level of liver enzymes and kidney factors when compared with the control group. ECEO displayed promising efficacy in parasite reduction in vitro and in the animal model. ECEO can thus be used as an alternative medicine to treat CL.

Promising natural products for the treatment of cutaneous leishmaniasis: A review of in vitro and in vivo studies.

Although there are available treatments for cutaneous leishmaniasis (CL), the drugs used are far from ideal, toxic, and costly, in addition to the challenge faced by the development of resistance. Plants have been used as a source of natural compounds with antileishmanial action. However, few have reached the market and become phytomedicines with registration in regulatory agencies. Difficulties related to the extraction, purification, chemical identification, efficacy, safety, and production in sufficient quantity for clinical studies, hinder the emergence of new effective phytomedicines against leishmaniasis. Despite the difficulties reported, the major research centers in the world see that natural products are a trend concerning the treatment of leishmaniasis. The present work consists of a literature review of articles with in vivo studies, covering the period from January 2011 to December 2022, providing an overview of promising natural products for CL treatment. The papers show encouraging antileishmanial action of natural compounds with reduced parasite load and lesion size in animal models, suggesting new strategies for the treatment of the disease. The results reported in this review show advances in using natural products as safe and effective formulations, which can stimulate clinical studies to establish clinical therapy. In conclusion, the information in this review article serves as a preliminary basis for establishing a therapeutic protocol for future clinical trials that can validate the safety and efficacy of natural compounds, providing the development of affordable and safe phytomedicines for the treatment of CL.

Antileishmanial, cellular mechanisms, and cytotoxic effects of green synthesized zinc nanoparticles alone and in combined with glucantime against Leishmania major infection.

BACKGROUND: We decided to investigate the antileishmanial, cellular mechanisms, and cytotoxic effects of green synthesized Zinc nanoparticles (ZnNPs) alone and combined with glucantime against Leishmania major infection. METHODS: The effect of green synthesized ZnNP on L. major amastigote was studied through macrophage cells. The mRNA expression level of iNOS and IFN-γ followed by the exposure of J774-A1 macrophage cells to ZnNPs was assessed by Real-time PCR. The Caspase-3-like activity of promastigotes exposed to ZnNPs was studied. Effects of ZnNPs alone and combined with glucantime (MA) were studied on cutaneous leishmaniasis in BALB/c mice. RESULTS: ZnNPs displayed the spherical shape with sizes ranging from 30 to 80 nm. The obtained IC50 values for ZnNPs, MA, and ZnNPs + MA were 43.2, 26.3, and 12.6 µg/mL, respectively; indicating the synergistic effects of ZnNPs in combination with MA. CL lesions had completely improved in the mice received with ZnNPs in combination with MA. The mRNA expression level of iNOS, TNF-α, and IFN-γ was dose-dependently (p < 0.01) upregulated; whereas it was downregulated in IL-10. ZnNPs markedly stimulated the caspase-3 activation with no significant toxicity on normal cells. CONCLUSION: Based on these in vitro and in vivo results, green synthesized ZnNPs, mainly along with MA, showed that has the potential to be introduced as a new drug for CL therapy. Triggering of NO production, and inhibition of infectivity rate are revealed as mechanisms of action ZnNPs on L. major. But, supplementary investigations are necessary to clear the efficacy and safety of these agents.

In vitro and in vivo antileishmanial activity of the hanging sedge flavonoids based on bio- guided fractionation assay.

As a major public health issue, cutaneous leishmaniasis (CL) has a number of complications, including drug resistance and poor response to conventional treatments. Over the last decade, research on natural sources for finding new antileishmanial agents has been a critical part of tropical disease research. Natural products also should be regarded as one of the most valuable applications for CL infection drug development. In this study, we assessed the in vitro and in vivo antileishmanial potential of Carex pendula Huds. (hanging sedge) methanolic extract and its fractions against Leishmania major produced cutaneous infection. Although the methanolic extract and its fractions exhibited suitable activity, the ethyl acetate fraction showed the best activity (with the half maximal inhibitory concentration IC50  = 1.627 ± 0.211 mg/mL). The toxicity and selectivity indices (SI) of all samples were determined in murine peritoneal macrophage cells (J774A.1) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. The flavonoid components of the ethyl acetate fraction were identified using liquid chromatography electrospray ionization mass spectrometry (LC-ESI MS/MS). Nine chemical compounds were identified in this fraction, including three flavonols, four flavanonols, and two flavan derivatives. L. major-infected mice were used as an in vivo model because the methanolic extract was effective against L. major promastigotes in the mammalian cell line J774A.1 with SI = 2.514 (tail lesion size model). In silico analysis of identified compounds also revealed a favorable interaction between compounds 2-5 and L. major protein targets (3UIB, 4JZX, 4JZB, 5L4N, and 5L42). According to the findings of this study, the ethyl acetate fraction (as flavonoid fraction) exhibited considerable in vitro antileishmanial activity.

The Therapeutic Effects of Curcumin-coated Gold Nanoparticle Against Leishmania Major Causative Agent of Zoonotic Cutaneous Leishmaniasis (ZCL): An In Vitro and In Vivo Study.

We synthesized and characterized curcumin-coated gold nanoparticles (Cur@AuNPs) and investigated their stability, cytotoxicity, leishmanicidal activity in in vitro and in in vivo experiments. Cur@AuNPs synthesized through a simple one-pot green chemistry technique. The in vitro leishmanicidal activity of curcumin-coated gold nanoparticles against extracellular promastigotes and intracellular amastigotes of protozoan parasite Leishmania major (L. major) was determined by applying the tetrazolium reduction colorimetric quantitative MTT technique. For in vivo assessment, the footpad lesion size and parasite burden in two infection site organs including lymph nodes and footpads of susceptible BALB/c mice infected with L. major were measured. Mice immune responses in all study groups were quantified by measuring the levels of gamma interferon (IFN-γ) and interleukin-4 (IL-4). Viability of Leishmania promastigotes significantly diminished with the inhibition in promastigotes growth (IC50) of 64.79 µg/mL and 29.89 µg/mL for 24 h and 48 h, respectively. In vitro nanoparticles treatment efficiently cleared the L. major amastigotes explanted in macrophages but had no harmful toxicity on the mice cells. In the in vivo condition, in the treated infected BALB/c mice the CL lesion size, Leishmania parasite burden, and IL-4 were decreased, while IFN-γ was significantly increased. The results suggest that Cur@AuNP was an effective compound against Leishmania parasite in vitro and in vivo, efficiently induced T-helper 1 (Th1) responses and augmented host cellular immune responses, and ending in a reduced Leishmania parasite burden. Therefore, it may be identified as a novel potential therapeutic approach for the local therapy of zoonotic CL treatment with high cure rates.

Preparation, optimization, and in vitro-in vivo evaluation of sorafenib-loaded polycaprolactone and cellulose acetate nanofibers for the treatment of cutaneous leishmaniasis.

The most common form of leishmaniasis is cutaneous leishmaniasis (CL). The major difficulties in the treatment of leishmaniasis include emergence of resistance, toxicity, long-term treatment, and the high cost of the current drugs. Although the therapeutic effect of sorafenib (SF) has been demonstrated in both in vitro and in vivo models of Leishmania infection, the therapeutic applications are limited due to severe drug-related toxicity; this is, in turn, due to non-specific distribution in the body. Thus, topical delivery has the advantage of the site directed delivery of SF. This research study evaluated SF-loaded hybrid nanofibers (NFs) which were composed of polycaprolactone (PCL) and cellulose acetate (CA) for the CL topical treatment. Accordingly, SF-loaded hybrid NFs were prepared using the electrospinning method. Formulation variables including total polymer concentration, drug/polymer ratio, and CA concentration were optimized using a full factorial design. The prepared SF-loaded NFs were then characterized for morphology, diameter, encapsulation efficiency (EE)%, drug loading (DL) %, and percentage of release efficiency during a 24-h period (RE24h%); the mechanical characteristics were also considered. The physical state of the drug in the optimized NF was evaluated by the X-ray diffraction analysis. Finally, its in vivo efficacy was determined in L. major-infected mice. The optimized formulation had a smooth, cylindrical, non-beaded shape fiber with a diameter of 281.44 nm, EE of 97.96%, DL of 7.48%, RE of 51.05%, ultimate tensile strength of 1.08 MPa, and Young's moduli of 74.96 MPa. The XRD analysis also demonstrated the amorphous state of SF in NF. Further, the in vivo results displayed the higher anti-leishmanial activity of the SF-loaded hybrid NF by efficiently healing lesion and successfully reducing the parasite burden. This, thus, indicated the potential of the clinical capability of the SF-loaded hybrid NF for the effective treatment of CL.

[An In Vitro Study on Sonodynamic Therapy of Leishmania tropica Using Curcumin]. / Kurkumin Kullanilarak Leishmania tropica'nin Sonodinamik Tedavisi Üzerine Bir In Vitro Çalisma.

Leishmaniasis is an infectious disease that is transmitted by Phlebotomus, 400 thousand new cases appearing every year, and approximately 350 million people are at risk, and accepted by the World Health Organization as one of the six important tropical diseases. Cutaneous leishmaniasis is a disease that occurs on exposed areas of the body and is characterized by long-term non-healing skin lesions. Although the treatment methods applied today vary according to the clinical picture of the patient, the immune system of the person and the causative agent Leishmania species, there is still no standard treatment scheme that has few side effects and can be used in the treatment of leishmaniasis. Therefore, alternative treatment methods with less side effects are being tried. Sonodynamic therapy (SDT) has also emerged as an active antimicrobial research area in recent years. SDT, a new modality for antibacterial therapy, aims to increase antibacterial effects with the simultaneous combination of low-intensity ultrasound and sonosensitizer. There is no information in the literature about the effect of SDT on parasites. In this study, it was aimed to demontrate the anti-leishmanial effect and possible mechanisms of curcumin mediated SDT on L.tropica promastigotes in vitro. Parasites were incubated with 0.25, 1.0, 4.0 and 15.6 micromolar (µM) of curcumin for one hour and subjected to 1 MHz frequency, 50% duty cycle and 3 W/cm2 intensity ultrasound irradiation. XTT assay was used to evaluate the viability of the cells and morphological changes were analyzed by Giemsa staining. Flow cytometry was used to quantify the fluorescence emitted by intracellular reactive oxygen species (ROS) signal, JC-1, cell cycle, Annexin V/PI staining reagents. With the combination of curcumin (15.6 µM) and ultrasound (3 W/cm2 intensity, seven minutes), L.tropica promastigote viability was found to be significantly decreased compared to the control group. Giemsa staining results showed that 15.6 µM curcumin mediated SDT induced several morphological alterations in L.tropica promastigotes typical for apoptosis. Late apoptosis was observed in 15.6 µM curcumin combined SDT treated parasites according to Annexin/PI staining. Besides, curcumin mediated SDT caused mitochondrial membrane potential (∆á´ªm) loss. Cell cycle analysis data indicated that curcumin based SDT caused an subG1 arrest in the cell cycle of L.tropica promastigotes. The generation of intracellular ROS detected by flow cytometry was increased in L.tropica promastigotes treated with curcumin mediated SDT. This study provided new data elucidating the molecular mechanism underlying the anti-leishmanial effect of curcumin mediated SDT. Curcumin mediated SDT has the potential to inactivate L.tropica promastigotes. However, further testing with amastigote or animal models is needed.

Antileishmanial Activities of Medicinal Herbs and Phytochemicals In Vitro and In Vivo: An Update for the Years 2015 to 2021.

Leishmaniasis is one of the most neglected tropical diseases that present areal public health problems worldwide. Chemotherapy has several limitations such as toxic side effects, high costs, frequent relapses, the development of resistance, and the requirement for long-term treatment. Effective vaccines or drugs to prevent or cure the disease are not available yet. Therefore, it is important to dissect antileishmanial molecules that present selective efficacy and tolerable safety. Several studies revealed the antileishmanial activity of medicinal plants. Several organic extracts/essential oils and isolated natural compounds have been tested for their antileishmanial activities. Therefore, the aim of this review is to update and summarize the investigations that have been undertaken on the antileishmanial activity of medicinal plants and natural compounds derived, rom plants from January 2015 to December 2021. In this review, 94 plant species distributed in 39 families have been identified with antileishmanial activities. The leaves were the most commonly used plant part (49.5%) followed by stem bark, root, and whole plant (21.9%, 6.6%, and 5.4%, respectively). Other plant parts contributed less (<5%). The activity was reported against amastigotes and/or promastigotes of different species (L. infantum, L. tropica, L. major, L. amazonensis, L. aethiopica, L. donovani, L. braziliensis, L. panamensis, L. guyanensis, and L. mexicana). Most studies (84.2%) were carried out in vitro, and the others (15.8%) were performed in vivo. The IC50 values of 103 plant extracts determined in vitro were in a range of 0.88 µg/mL (polar fraction of dichloromethane extract of Boswellia serrata) to 98 µg/mL (petroleum ether extract of Murraya koenigii). Among the 15 plant extracts studied in vivo, the hydroalcoholic leaf extract of Solanum havanense reduced parasites by 93.6% in cutaneous leishmaniasis. Voacamine extracted from Tabernaemontana divaricata reduced hepatic parasitism by ≈30 times and splenic parasitism by ≈15 times in visceral leishmaniasis. Regarding cytotoxicity, 32.4% of the tested plant extracts against various Leishmania species have a selectivity index higher than 10. For isolated compounds, 49 natural compounds have been reported with anti-Leishmania activities against amastigotes and/or promastigotes of different species (L. infantum, L. major, L. amazonensis, L. donovani and L. braziliensis). The IC50 values were in a range of 0.2 µg/mL (colchicoside against promastigotes of L. major) to 42.4 µg/mL (dehydrodieuginol against promastigotes of L. amazonensis). In conclusion, there are numerous medicinal plants and natural compounds with strong effects (IC50 < 100 µg/mL) against different Leishmania species under in vitro and in vivo conditions with good selectivity indices (SI > 10). These plants and compounds may be promising sources for the development of new drugs against leishmaniasis and should be investigated in randomized clinical trials.

Meglumine antimoniate was associated with a higher cure rate than liposomal amphotericin B in the treatment of American tegumentary leishmaniasis: A retrospective cohort study from a Leishmania braziliensis-endemic area.

Background: Pentavalent antimonials (PAs) are the primary therapeutic option for American tegumentary leishmaniasis (ATL). However, the use of these drugs is complicated by adverse events (AEs), resistance and contraindications. Alternative therapies relative effectiveness is not well established. Objective: This study compared the effectiveness of liposomal amphotericin B (LAB) with intravenous meglumine antimoniate (NMG) in the treatment of ATL. We also analysed and compared associated AEs and treatment interruption rates. Methods: This was a retrospective cohort study from Brazil. The potential risk factors for the primary outcome were age, sex, total cutaneous lesion area, presence of mucosal lesions, AEs and treatment interruption. The primary outcome was lesion healing within 6 months of treatment. AEs and treatment interruption were also analysed. Multiple analytic strategies were employed to evaluate the reliability of the results. Results: Before propensity score (PS) matching, patients in the LAB group were older and had a higher frequency of mucosal lesions. The NMG group had a higher cure rate than the LAB group (cure rate 88% versus 55% respectively) in the adjusted analysis (relative risk (RR)=1.55 95% CI: 1.19 - 2.02) and after PS matching (RR=1.63 95% CI: 1.20 - 2.21). NMG group had a higher AE rate (event rate 52% versus 44%) in the adjusted analysis (RR= 1.61, 95% CI: 1.06 - 2.43, p=0.02), but this result was not observed after PS matching (RR= 0.87, 95% CI: 0.49 -1.52, p= 0.61). Conclusions: We observed that the NMG group had a higher cure rate than the LAB group, with an equivocally higher EV rate in the adjusted analysis.

Stachys lavandulifolia Vahl. exhibits promising in vitro and in vivo antileishmanial activity against Leishmania major infection.

This study aimed to consider the in vitro and in vivo effects of the Stachys lavandulifolia methanolic extract (SLME) (2.5, 5, 10, 25, 50, 100 µg/mL) against Leishmania major infection. The in vitro antileishmanial effects of SLME was studies on promastigote and amastigote forms of L. major. The effect of SLME on the nitric oxide (NO) and apoptosis, secretion of Th1/2 cytokines, and infectivity rate in macrophages cells were also studies. The cytotoxicity of SLME on human (THP-1) and murine (J774-A1 cell) macrophage cells was investigated through the measuring the 50% cytotoxic concentrations (CC50). Moreover, the in vivo effects of SLME for healing the cutaneous leishmaniasis (CL) lesions in infected BALB/c mice studied by assessing the lesions size and the parasite load during four weeks of treatment. The calculated 50% inhibitory concentration (IC50) valuesfor SLME and meglumine antimoniate (MA) against the promastigote stage were 23.4 and 71.1 µg/mL, respectively. For amastigote stage, the IC50 values for SLME and MA were 39.3 µg/mL and 44.3 µg/mL, respectively. Followed by 28 days' topically therapy with SLME at doses of 50 and 100 mg/kg/day, the CL lesions size as well as parasite load were significantly (p<0.001) reduced; such that the recovery percentage of the infected mice was 80% and 97% after treatment with SLME at the dose of 50 and 100 mg/kg, respectively. SLME also markedly induced the NO production and apoptosis; whereas decreased infection rate in macrophage cells. After incubation of infected macrophages with SLME, the level interferon gamma was meaningfully (p<0.001) elevated as a dose-dependent response; in contrast, release of interleukin 10 (IL-10) and IL-4 markedly (p<0.001) decreased. The CC50 value for SLME against THP-1 and J774-A1 cell was 996.4 µg/mL and 741.3 µg/mL, respectively. The calculated selectivity index of >10 for SLME and MA confirmed their specificity to amastigotes and the low toxicity for macrophages. Our results showed the potent effects of SLME in eliminating and controlling Leishmania parasites in both in vitro and in vivo assays. Based on the current experimental study, SLME can be suggested as an alternative medicine for the isolation and production of a new agent for treating CL caused by L. major. Although, we found some cellular mechanisms of SLME against Leishmania parasites, but, additional surveys are necessary to specify the accurate mechanisms of action, toxicity, and its efficacy mainly in human subjects.

Exploring the Immunotherapeutic Potential of Oleocanthal against Murine Cutaneous Leishmaniasis.

Leishmaniasis is a major tropical disease with increasing global incidence. Due to limited therapeutic options with severe drawbacks, the discovery of alternative treatments based on natural bioactive compounds is important. In our previous studies we have pointed out the antileishmanial activities of olive tree-derived molecules. In this study, we aimed to investigate the in vitro and in vivo antileishmanial as well as the in vivo immunomodulatory effects of oleocanthal, a molecule that has recently gained increasing scientific attention. Pure oleocanthal was isolated from extra virgin olive oil through extraction and chromatography techniques. The in vitro antileishmanial effects of oleocanthal were examined with a resazurin-based assay, while its in vivo efficacy was evaluated in Leishmania major-infected BALB/c mice by determining footpad induration, parasite load in popliteal lymph nodes, histopathological outcome, antibody production, cytokine profile of stimulated splenocytes and immune gene expression, at three weeks after the termination of treatment. Oleocanthal demonstrated in vitro antileishmanial effect against both L. major promastigotes and intracellular amastigotes. This effect was further documented in vivo as demonstrated by the suppressed footpad thickness, the decreased parasite load and the inflammatory cell influx at the infection site. Oleocanthal treatment led to the dominance of a Th1-type immunity linked with resistance against the disease. This study establishes strong scientific evidence for olive tree-derived natural products as possible antileishmanial agents and provides an adding value to the scientific research of oleocanthal.

PHARMACOLOGICAL AND MECHANISTIC STUDIES OF UREQUINONA, A MOLECULE FROM ROOTS OF PENTALINON ANDRIEUXII MUELL-ARG THAT HEALS MURINE LEISHMANIA MEXICANA INFECTION.

In this work we tested both the in vitro and in vivo anti-Leishmania mexicana activity of a molecule we originally identified in the root of Pentalinon andrieuxii Muell-Arg, a plant that is widely used in Mayan traditional medicine. The chemical name of this molecule is 24-methylcholesta-4-24(28)-dien-3-one, but for simplicity's sake, we assigned the short and trivial name of urequinona that will be used throughout this work. It induces necrosis and apoptosis of promastigotes cultured in vitro and extensive ultrastructural damage of amastigotes. It also induces production of Interleukin (IL)-2 and interferon (IFN)-γ by splenic cells from infected and urequinona treated mice stimulated in vitro with parasite antigen (Ag) but inhibits the production of IL-6 and IL-12p70 by bone-marrow-derived macrophages (BMM) infected in vitro and then treated with urequinona. It also induces activation of transcription factors such as NFkB and AP-1 (NFkB/AP-1) in RAW reporter cells. We also developed a novel pharmaceutical preparation of urequinona encapsulated in hydroxyethyl cellulose for dermal application that significantly reduced (P < 0.05) experimentally induced ear lesions of C57BL/6 mice. We conclude the preparation containing this molecule is a good candidate for a novel anti-leishmanial drug's preparation.

Therapeutic effects of Lucilia sericata larval excretion/secretion products on Leishmania major under in vitro and in vivo conditions.

BACKGROUND: Leishmaniasis is a neglected infectious disease caused by protozoa of the genus Leishmania. The disease generally manifests as characteristic skin lesions which require lengthy treatment with antimonial drugs that are often associated with adverse side effects. Therefore, a number of studies have focused on natural compounds as promising drugs for its treatment. This study aimed to evaluate the effects of larval excretion/secretion products (ES) of Lucilia sericata in crude and fractionated forms on Leishmania major, by using in vitro and in vivo models. METHODS: The in vitro experiments involved evaluation of ES on both promastigotes and macrophage-engulfed amastigotes, whereas the in vivo experiments included comparative treatments of skin lesions in L. major-infected mice with Eucerin-formulated ES and Glucantime. RESULTS: The half maximal inhibitory concentrations of the crude ES, > 10-kDa ES fraction, < 10-kDa ES fraction, and Glucantime were 38.7 µg/ml, 47.6 µg/ml, 63.3 µg/ml, and 29.1 µg/ml, respectively. Significant differences were observed between percentage viabilities of promastigotes treated with the crude ES and its fractions compared with the negative control (P < 0.0001). The crude ES was more effective on amastigotes than the two ES fractions at 300 µg/ml. The macroscopic measurements revealed that the reduction of lesion size in mice treated with the crude ES followed quicker cascades of healing than that of mice treated with Glucantime and the ES fractions. CONCLUSIONS: The present study showed that the larval ES of L. sericata in both crude and fractionated forms are effective for both intracellular and extracellular forms of L. major. Also, the ES exert both topical and systemic effects on mice experimentally infected with L. major.

Directrices para el tratamiento de las leishmaniasis en la región de las américas - 2 ed / Guidelines for the treatment of leishmaniasis in the region of the Americas - 2 ed

Las leishmaniasis son enfermedades infecciosas desatendidas de gran importancia en la Región de las Américas debido a su morbilidad, mortalidad y amplia distribución geográfica. De las tres formas clínicas principales, la cutánea es la más común y la visceral es la forma más grave, ya que puede causar la muerte de hasta 90% de las personas que no reciban tratamiento. En el 2013, la Organización Panamericana de la Salud (OPS) elaboró recomendaciones para el tratamiento de las leishmaniasis en la Región de las Américas utilizando la metodología de clasificación de la valoración, la elaboración y la evaluación de las recomendaciones (GRADE, por su sigla en inglés). No obstante, dada la evidencia acumulada desde entonces, se hizo necesario revisar esas recomendaciones. En esta segunda edición se presentan las recomendaciones actualizadas sobre el tratamiento de las leishmaniasis, y se detallan los esquemas y los criterios de indicación del tratamiento en el contexto regional. Estas directrices presentan modificaciones sustanciales con respecto a la primera edición. En el caso de la leishmaniasis cutánea, se ha eliminado el ketoconazol de las opciones terapéuticas, el número de especies de Leishmania para las que hay evidencia sólida de la eficacia de la miltefosina ha aumentado de dos a cuatro y la recomendación de administrar antimoniales intralesionales ahora es fuerte. Con respecto a la leishmaniasis mucosa, se incluye una recomendación fuerte sobre el uso de antimoniales pentavalentes con o sin pentoxifilina oral. Por lo que respecta a la leishmaniasis visceral, la recomendación fuerte sobre el uso de antimoniales pentavalentes y desoxicolato de anfotericina B ahora es condicional. También hay evidencia contundente en contra del uso de miltefosina en pacientes con leishmaniasis causada por Leishmania infantum. Otros cambios importantes son el desglose de las recomendaciones según si se trata de pacientes adultos o pediátricos, la inclusión de las especies de Leishmania y, en el caso de los pacientes inmunocomprometidos, la introducción de una recomendación fuerte contra el uso de antimoniales pentavalentes. Esta segunda edición es una versión revisada de la publicación Leishmaniasis en las Américas: recomendaciones para el tratamiento: https://iris.paho.org/handle/10665.2/7704

Involvement of tryparedoxin peroxidase (TryP) and trypanothione reductase (TryR) in antimony unresponsive of Leishmania tropica clinical isolates of Iran.

Clinical resistance to pentavalent antimonial compounds has long been recognized as a major problem in the treatment of human leishmaniasis. Trypanothione metabolism, the main form of thiol, has shown to play a central role in antimony resistance of laboratory-generated resistant Leishmania spp. and field-isolated resistant L. donovani; but the mechanism of antimony resistance in the clinical isolates of L. tropica causing anthroponotic cutaneous leishmaniasis (ACL) is less studied. Patients were selected among confirmed positive ACL cases who referred to Pasteur Institute of Iran, Tehran, from endemic regions of north-east and south of Iran. L. tropica clinical isolates were collected from patients who were either treatment-responsive (MAS=S1 to S5) or unresponsive (MAR=R1 to R4) to Glucantime® (meglumine antimoniate=MA). Isolates were tested for sensitivity to trivalent antimony (SbIII) in promastigotes and to pentavalent antimony (SbV) in intracellular amastigotes stages. Intracellular thiol levels were assayed and trypanothione-dependent components, including trypanothione reductase (TR) and tryparedoxin peroxidase I (TryP) were analysed at protein level and enzymatic activity in isolates. The MAR isolates had an approximate two fold increase in the levels of intracellular thiols (P< 0.05) accompanied by an average 5-10 fold increase in in vitro resistance to antimony. TryP was amplified at the protein level in all MAR strains as compared to the MAS strains (range: 2.8-5.6 fold). All MAR isolates metabolized H2O2 at higher rates than MAS isolates (8.55±0.75 nmol/min/mg vs. 3.14±0.36 nmol/min/mg) (P< 0.05). In addition, levels of TryR protein were also markedly elevated in 3 out of 4 MAR isolates (range: 2.2-4.1 fold). This was accompanied by overexpressed TryR activity (mean level of 46.83±2.43 for extracts of MAR vs. 20.98±3.02 for MAS strains) (P< 0.05). Elevated levels of TryP, active enzyme in peroxide detoxification, were observed in MAR parasites resulting in an increased metabolism of H2O2. TryR activity was overexpressed on average in extracts of MAR strains, but not in all isolates. Enhanced anti-oxidant defenses through thiol metabolism may play a significant role in clinical resistance of ACL patients to Glucantime.