Piperine, quercetin, and curcumin identified as promising natural products for topical treatment of cutaneous leishmaniasis.

Leishmania braziliensis (L. braziliensis) causes cutaneous leishmaniasis (CL) in the New World. The costs and the side effects of current treatments render imperative the development of new therapies that are affordable and easy to administer. Topical treatment would be the ideal option for the treatment of CL. This underscores the urgent need for affordable and effective treatments, with natural compounds being explored as potential solutions. The alkaloid piperine (PIP), the polyphenol curcumin (CUR), and the flavonoid quercetin (QUE), known for their diverse biological properties, are promising candidates to address these parasitic diseases. Initially, the in vitro cytotoxicity activity of the compounds was evaluated using U-937 cells, followed by the assessment of the leishmanicidal activity of these compounds against amastigotes of L. braziliensis. Subsequently, a golden hamster model with stationary-phase L. braziliensis promastigote infections was employed. Once the ulcer appeared, hamsters were treated with QUE, PIP, or CUR formulations and compared to the control group treated with meglumine antimoniate administered intralesionally. We observed that the three organic compounds showed high in vitro leishmanicidal activity with effective concentrations of less than 50 mM, with PIP having the highest activity at a concentration of 8 mM. None of the compounds showed cytotoxic activity for U937 macrophages with values between 500 and 700 mM. In vivo, topical treatment with QUE daily for 15 days produced cured in 100% of hamsters while the effectiveness of CUR and PIP was 83% and 67%, respectively. No failures were observed with QUE. Collectively, our data suggest that topical formulations mainly for QUE but also for CUR and PIP could be a promising topical treatment for CL. Not only the ease of obtaining or synthesizing the organic compounds evaluated in this work but also their commercial availability eliminates one of the most important barriers or bottlenecks in drug development, thus facilitating the roadmap for the development of a topical drug for the management of CL caused by L. braziliensis.

Leishmania braziliensis enhances monocyte responses to promote anti-tumor activity.

Innate immune cells can undergo long-term functional reprogramming after certain infections, a process called trained immunity (TI). Here, we focus on antigens of Leishmania braziliensis, which induced anti-tumor effects via trained immunity in human monocytes. We reveal that monocytes exposed to promastigote antigens of L. braziliensis develop an enhanced response to subsequent exposure to Toll-like receptor (TLR)2 or TLR4 ligands. Mechanistically, the induction of TI in monocytes by L. braziliensis is mediated by multiple pattern recognition receptors, changes in metabolism, and increased deposition of H3K4me3 at the promoter regions of immune genes. The administration of L. braziliensis exerts potent anti-tumor capabilities by delaying tumor growth and prolonging survival of mice with non-Hodgkin lymphoma. Our work reveals mechanisms of TI induced by L. braziliensis in vitro and identifies its potential for cancer immunotherapy.

The perfect storm: Cutaneous leishmaniasis in troops deployed in the Atlantic forest of Argentina.

American cutaneous leishmaniasis (ACL) is the most prevalent form of leishmaniasis, associated with an ulcerative and stigmatizing mucocutaneous pathology. This study assessed the incidence of Leishmania (Viannia) braziliensis in members of the Argentine Army who were exposed to sandfly bites in Iguazú National Park (INP), northeastern Argentina, during an outbreak of ACL in 2019, and the presence of Leishmania in rodents, opossums and phlebotomine sandflies collected in the area of exposure. Samples from military personnel, wild animals and phlebotomine sandflies were analysed. A total of 20 (40%) patients among the Army personnel and two Akodon montensis rodents (11%) were positive for the presence of Leishmania sp. genes by PCR, while Nyssomyia whitmani and Migonemyia migonei, competent vectors of Leishmania, were also found at the same site. Sequences of hsp70 DNA fragments obtained from human samples confirmed the identity of L. (V.) braziliensis. The risk to which military personnel carrying out activities in the forest are exposed is highlighted, and this risk extends to any worker and visitor who circulates without protection in the INP, coming into contact with transmission "hot spots" due to the concentration of vectors, reservoirs and/or parasites.

Leishmania (Leishmania) infantum DNA detection in Nyssomyia neivai in Vale do Ribeira, Paraná, Brazil.

BACKGROUND: The incidence of visceral leishmaniasis (VL) has increased in the Southern region of Brazil in recent years, especially in the State of Paraná. New species have been suggested with potential to act as vector in VL endemic areas. OBJECTIVES: Identify the Leishmania species in sand fly specimens collected from 2016 to 2018 in the municipality of Itaperuçu, Vale do Ribeira, Paraná, Brazil. METHODS: Light traps were used for collections and for the analysis of sand fly were used the multiplex polymerase chain reaction (PCR) methodology and subsequent sequencing. FINDINGS: Among the collected specimens, 88.62% were attributed to the species Nyssomyia neivai, which were grouped into 176 pools. Three positive pools were detected: two with Leishmania (Viannia) braziliensis and one with L. (Leishmania) infantum. The positivity rate for the parasite was 0.25% based on the presence of at least one infected insect in the pool. MAIN CONCLUSIONS: The detection of L. infantum in Ny. neivai draws attention due to its abundance and anthropophily in the State of Paraná. Moreover, this finding is considered as an alert and suggests that the vector competence of Ny. neivai and the criteria for its incrimination should be carried out, given its wide distribution in southern of Brazil.

Disseminated Leishmaniasis, a Severe Form of Leishmania braziliensis Infection.

Disseminated leishmaniasis (DL) is an emergent severe disease manifesting with multiple lesions. To determine the relationship between immune response and clinical and therapeutic outcomes, we studied 101 DL and 101 cutaneous leishmaniasis (CL) cases and determined cytokines and chemokines in supernatants of mononuclear cells stimulated with leishmania antigen. Patients were treated with meglumine antimoniate (20 mg/kg) for 20 days (CL) or 30 days (DL); 19 DL patients were instead treated with amphotericin B, miltefosine, or miltefosine and meglumine antimoniate. High levels of chemokine ligand 9 were associated with more severe DL. The cure rate for meglumine antimoniate was low for both DL (44%) and CL (60%), but healing time was longer in DL (p = 0.003). The lowest cure rate (22%) was found in DL patients with >100 lesions. However, meglumine antimoniate/miltefosine treatment cured all DL patients who received it; therefore, that combination should be considered as first choice therapy.

Short communication: The miR-155a-5p is correlated with increased ROS and impaired apoptosis in macrophages infected by Leishmania braziliensis.

Cutaneous leishmaniasis (CL) caused by Leishmania braziliensis, is a disease characterized by well-limited ulcerated lesions with raised borders in exposed parts of the body. miRNAs are recognized for their role in the complex and plastic interaction between host and pathogens, either as part of the host's strategy to neutralize infection or as a molecular mechanism employed by the pathogen to modulate host inflammatory pathways to remain undetected. The mir155 targets a broad range of inflammatory mediators, following toll-like receptors (TLRs) signaling. In this work, we evaluated the effects of the expression of miR155a-5p in human macrophages infected with L. braziliensis. Our results show that miR155a-5p is inversely correlated with early apoptosis and conversely, seems to influence an increment in the oxidative burst in these cells. Altogether, we spotted a functional role of the miR155a-5p in CL pathogenesis, raising the hypothesis that an increased miR-155 expression by TLR ligands influences cellular mechanisms settled to promote both killing and control of parasite density after infection.

The protein map of the protozoan parasite Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis and Leishmania (Leishmania) infantum during growth phase transition and temperature stress.

Leishmania parasites cause a spectrum of diseases termed leishmaniasis, which manifests in two main clinical forms, cutaneous and visceral leishmaniasis. Leishmania promastigotes transit from proliferative exponential to quiescent stationary phases inside the insect vector, a relevant step that recapitulates early molecular events of metacyclogenesis. During the insect blood meal of the mammalian hosts, the released parasites interact initially with the skin, an event marked by temperature changes. Deep knowledge on the molecular events activated during Leishmania-host interactions in each step is crucial to develop better therapies and to understand the pathogenesis. In this study, the proteomes of Leishmania (Leishmania) amazonensis (La), Leishmania (Viannia) braziliensis (Lb), and Leishmania (Leishmania) infantum (syn L. L. chagasi) (Lc) were analyzed using quantitative proteomics to uncover the proteome modulation in three different conditions related to growth phases and temperature shifts: 1) exponential phase (Exp); 2) stationary phase (Sta25) and; 3) stationary phase subjected to heat stress (Sta34). Functional validations were performed using orthogonal techniques, focusing on α-tubulin, gp63 and heat shock proteins (HSPs). Species-specific and condition-specific modulation highlights the plasticity of the Leishmania proteome, showing that pathways related to metabolism and cytoskeleton are significantly modulated from exponential to stationary growth phases, while protein folding, unfolded protein binding, signaling and microtubule-based movement were differentially altered during temperature shifts. This study provides an in-depth proteome analysis of three Leishmania spp., and contributes compelling evidence of the molecular alterations of these parasites in conditions mimicking the interaction of the parasites with the insect vector and vertebrate hosts. SIGNIFICANCE: Leishmaniasis disease manifests in two main clinical forms according to the infecting Leishmania species and host immune responses, cutaneous and visceral leishmaniasis. In Brazil, cutaneous leishmaniasis (CL) is associated with L. braziliensis and L. amazonensis, while visceral leishmaniasis, also called kala-azar, is caused by L. infantum. Leishmania parasites remodel their proteomes during growth phase transition and changes in their mileu imposed by the host, including temperature. In this study, we performed a quantitative mass spectrometry-based proteomics to compare the proteome of three New world Leishmania species, L. amazonensis (La), L. braziliensis (Lb) and L. infantum (syn L. chagasi) (Lc) in three conditions: a) exponential phase at 25 °C (Exp); b) stationary phase at 25 °C (Sta25) and; c) stationary phase subjected to temperature stress at 34 °C (Sta34). This study provides an in-depth proteome analysis of three Leishmania spp. with varying pathophysiological outcomes, and contributes compelling evidence of the molecular alterations of these parasites in conditions mimicking the interaction of the parasites with the insect vector and vertebrate hosts.

Consolidation of a Molecular Signature of Healing in Cutaneous Leishmaniasis Is Achieved during the First 10 Days of Treatment.

The immune response is central to the pathogenesis of cutaneous leishmaniasis (CL). However, most of our current understanding of the immune response in human CL derives from the analysis of systemic responses, which only partially reflect what occurs in the skin. In this study, we characterized the transcriptional dynamics of skin lesions during the course of treatment of CL patients and identified gene signatures and pathways associated with healing and nonhealing responses. We performed a comparative transcriptome profiling of serial skin lesion biopsies obtained before, in the middle, and at the end of treatment of CL patients (eight who were cured and eight with treatment failure). Lesion transcriptomes from patients who healed revealed recovery of the stratum corneum, suppression of the T cell-mediated inflammatory response, and damping of neutrophil activation, as early as 10 d after initiation of treatment. These transcriptional programs of healing were consolidated before lesion re-epithelization. In stark contrast, downregulation of genes involved in keratinization was observed throughout treatment in patients who did not heal, indicating that in addition to uncontrolled inflammation, treatment failure of CL is mediated by impaired mechanisms of wound healing. This work provides insights into the factors that contribute to the effective resolution of skin lesions caused by Leishmania (Viannia) species, sheds light on the consolidation of transcriptional programs of healing and nonhealing responses before the clinically apparent resolution of skin lesions, and identifies inflammatory and wound healing targets for host-directed therapies for CL.

Big is not better: Comparing two alpha-Gal-bearing glycotopes in neoglycoproteins as biomarkers for Leishmania (Viannia) braziliensis infection.

The protozoan parasite Leishmania (Viannia) braziliensis is among Latin America's most widespread Leishmania species and is responsible for tegumentary leishmaniasis (TL). This disease has multiple clinical presentations, with cutaneous leishmaniasis (CL) being the most frequent. It manifests as one or a few localized skin ulcers, which can spread to other body areas. Hence, early diagnosis and treatment, typically with pentavalent antimonials, is critical. Traditional diagnostic methods, like parasite culture, microscopy, or the polymerase chain reaction (PCR) for detection of the parasite DNA, have limitations due to the uneven distribution of parasites in biopsy samples. Nonetheless, studies have revealed high levels of parasite-specific anti-α-Gal antibodies in L. (V.) braziliensis-infected patients. Previously, we demonstrated that the neoglycoprotein NGP28b, consisting of the L. (Leishmania) major type-2 glycoinositolphospholipid (GIPL)-3-derived trisaccharide Galpα1,6Galpα1,3Galfß conjugated to bovine serum albumin (BSA) via a linker, acts as a reliable serological biomarker (BMK) for L. (V.) braziliensis infection in Brazil. This indicates the presence of GIPL-3 or a similar structure in this parasite, and its terminal trisaccharide either functions as or is part of an immunodominant glycotope. Here, we explored whether extending the trisaccharide with a mannose unit would enhance its efficacy as a biomarker for the serological detection of L. (V.) braziliensis. We synthesized the tetrasaccharide Galpα1,6Galpα1,3Galfß1,3Manpα(CH2)3SH (G31SH) and conjugated it to maleimide-functionalized BSA to afford NGP31b. When we assessed the efficacy of NGP28b and NGP31b by chemiluminescent enzyme-linked immunosorbent assay on a cohort of CL patients with L. (V.) braziliensis infection from Bolivia and Argentina against a healthy control group, both NGPs exhibited similar or identical sensitivity, specificity, and accuracy. This finding implies that the mannose moiety at the reducing end is not part of the glycotope recognized by the parasite-specific anti-α-Gal antibodies in patients' sera, nor does it exert a relevant influence on the terminal trisaccharide's conformation. Moreover, the mannose does not seem to inhibit glycan-antibody interactions. Therefore, NGP31b is a viable and dependable BMK for the serodiagnosis of CL caused by L. (V.) braziliensis.

Interaction between diterpene icetexanes and old yellow enzymes of Leishmania braziliensis and Trypanosoma cruzi.

Old Yellow Enzymes (OYEs) are flavin-dependent redox enzymes that promote the asymmetric reduction of activated alkenes. Due to the high importance of flavoenzymes in the metabolism of organisms, the interaction between OYEs from the parasites Trypanosoma cruzi and Leishmania braziliensis and three diterpene icetexanes (brussonol and two analogs), were evaluated in the present study, and differences in the binding mechanism and inhibition capacity of these molecules were examined. Although the aforementioned compounds showed poor and negligible activities against T. cruzi and L. braziliensis cells, respectively, the experiments with the purified enzymes indicated that the interaction occurs by divergent mechanisms. Overall, the ligands' inhibitory effect depends on their accessibility to the N5 position of the flavin's isoalloxazine ring. The results also indicated that the OYEs found in both parasites share structural similarities and showed affinities for the diterpene icetexanes in the same range. Nevertheless, the interaction between OYEs and ligands is directed by enthalpy and/or entropy in distinct ways. In conclusion, the binding site of both OYEs exhibits remarkable plasticity, and a large range of different molecules, including that can be substrates and inhibitors, can bind this site. This plasticity should be considered in drug design using OYE as a target.

Anti-Leishmania compounds can be screened using Leishmania spp. expressing red fluorescence (tdTomato).

The main challenges associated with leishmaniasis chemotherapy are drug toxicity, the possible emergence of resistant parasites, and a limited choice of therapeutic agents. Therefore, new drugs and assays to screen and detect novel active compounds against leishmaniasis are urgently needed. We thus validated Leishmania braziliensis (Lb) and Leishmania infantum (Li) that constitutively express the tandem tomato red fluorescent protein (tdTomato) as a model for large-scale screens of anti-Leishmania compounds. Confocal microscopy of Lb and Li::tdTomato revealed red fluorescence distributed throughout the entire parasite, including the flagellum, and flow cytometry confirmed that the parasites emitted intense fluorescence. We evaluated the infectivity of cloned promastigotes and amastigotes constitutively expressing tdTomato, their growth profiles in THP-1 macrophages, and susceptibility to trivalent antimony, amphotericin, and miltefosine in vitro. The phenotypes of mutant and wild-type parasites were similar, indicating that the constitutive expression of tdTomato did not interfere with the evaluated parameters. We applied our validated model to a repositioning strategy and assessed the susceptibility of the parasites to eight commercially available drugs. We also screened 32 natural plant and fungal extracts and 10 pure substances to reveal new active compounds. The infectivity and Glucantime treatment efficacy of BALB/c mice and golden hamsters infected with Lb and Li::tdTomato mutant lines, respectively, were very similar compared to animals infected with wild-type parasites. Standardizing our methodology would offer more rapid, less expensive, and easier assays to screen of compounds against L. braziliensis and L. infantum in vitro and in vivo. Our method could also enhance the discovery of active compounds for treating leishmaniasis.

Novel detection of Leishmania RNA virus-1 (LRV-1) in clinical isolates of Leishmania Viannia panamensis.

American tegumentary leishmaniasis comprises a discrete set of clinical presentations endemic to Latin America. Leishmania RNA virus-1 (LRV-1) is a double-stranded RNA virus identified in 20­25% of the Leishmania Viannia braziliensis and L. V. guyanensis, however not in L. V. panamensis. This is the first report of LRV-1 in L. V. panamensis and its associations with clinical phenotypes of ATL. Unique surplus discard clinical isolates of L. V. panamensis were identified from the Public Health Ontario Laboratory (PHOL) and the Leishmania Clinic of the Instituto de Medicina Tropical 'Alexander von Humboldt' between 2012 and 2019 and screened for LRV-1 by real-time polymerase chain reaction. Patient isolates were stratified according to clinical phenotype. Of 30 patients with L. V. panamensis, 14 (47%) and 16 (53%) patients had severe and non-severe ATL, respectively. Five (36%) of 14 severe cases and 2 (12%) of 16 non-severe cases were positive for LRV-1, respectively. No differences in sex were observed for clinical phenotype and LRV-1 status. Although an association between LRV-1 status and clinical phenotype was not demonstrated, this is the first description of the novel detection of LRV-1 in L. V. panamensis, a species that has been documented predominantly in Central America.

Detection of Internal Transcribed Spacer 1 and hsp70 Genetic Markers Using Restriction Fragment Length Polymorphisms and Sequencing in Identification of Leishmania Species Causing Tegumentary Leishmaniasis in Brazil.

The identification of Leishmania species that cause tegumentary leishmaniasis (TL) is important for taxonomic and prognostic purposes. Molecular analysis using different Leishmania genomic targets is the most useful method for identifying Leishmania species. Therefore, we evaluated the performance of ribosomal RNA internal transcribed spacer 1 (ITS1) and heat shock protein (hsp70) genetic markers by polymerase chain reaction (PCR), followed by restriction fragment length polymorphism analysis (RFLP) and sequencing, for identification of Leishmania species. Samples from 84 Brazilian patients were amplified. Internal transcribed spacer 1 PCR followed by RFLP (HaeIII) [ITS1-RFLP (HaeIII)] identified 46.4% (39/84) of the samples as compatible with the Viannia subgenus. Internal transcribed spacer 1 PCR followed by sequencing (ITS1-sequencing) identified Leishmania (Viannia) braziliensis in 91.7% (77/84) of the TL samples, Leishmania (Leishmania) amazonensis in 3.6% (3/84), L. (V.) guyanensis in 2.4% (2/84), and L. (L.) infantum in 1.2% (1/84). One of the samples showed the same proportion of similarity with L. (V.) guyanensis and L. (V.) panamensis. hsp70 nested PCR followed by RFLP (HaeIII) [nested hsp70-RFLP (HaeIII)] identified 91.7% (77/84) of the samples as compatible with L. (V.) braziliensis/L. (V.) naiffi, 3.6% (3/84) with L. (L.) amazonensis, 1.2% (1/84) with L. (L.) infantum, and 3.6% (3/84) with L. (V.) guyanensis. hsp70 PCR followed by sequencing (hsp70-sequencing) identified L. (V.) braziliensis in 91.7% (77/84) of the TL samples, L. (L.) amazonensis in 3.6% (3/84), L. (V.) guyanensis in 3.6% (3/84), and L. (L.) infantum in 1.2% (1/84). Our findings clearly showed that nested hsp70-RFLP (HaeIII) is better than ITS1-RFLP (HaeIII) and that ITS1 or hsp70 PCR followed by sequencing was adequate for identifying Leishmania species. We also found that Leishmania (Viannia) braziliensis is the most common species causing TL in Brazil. Therefore, sequencing multiple target genes such as ITS1 and hsp 70 is more accurate than RFLP for identifying Leishmania species.

Chlorella vulgaris extract and Imiquimod as new therapeutic targets for leishmaniasis: An immunological approach.

The therapeutic regimen for the treatment of American Tegumentary Leishmaniasis (ATL) is targeted at the death of the parasite; therefore, it is essential to develop a treatment that can act on the parasite, combined with the modulation of the inflammatory profile. Thus, the aim of this study was to make an in vitro evaluation of the therapeutic potential of Chlorella vulgaris extract (CV) and Imiquimod for ATL. Selectivity indices (SI) were determined by inhibitory concentration assays (IC50) in L. braziliensis cells and cytotoxic concentrations (CC50) were measured in human cells using the MTT method, based on the CV microalgae extract (IC50 concentrations of 15.63 to 500 µg/mL; CC50 concentrations of 62.5-1000 µg/mL) in comparison with the reference drugs and Imiquimod. The immune response was evaluated in healthy human cells by gene expression (RT-qPCR) and cytokine production (Flow Cytometry). The CV extract (SI = 6.89) indicated promising results by showing higher SI than meglumine antimoniate (SI = 3.44) (reference drug). In all analyses, CV presented a protective profile by stimulating the production of Th1 profile cytokines to a larger extent than the reference drugs. Imiquimod showed a high expression for Tbx21, GATA3, RORc and Foxp3 genes, with increased production only of the TNF cytokine. Therefore, the data highlight the natural extract and Imiquimod as strong therapeutic or adjuvant candidates against ATL, owing to modulation of immune response profiles, low toxicity in human cells and toxic action on the parasite.

Assessing reservoir host status in leishmaniasis with special reference to the infectiousness of Leishmania (Viannia) braziliensis infections in wild rodents.

Molecular methods have been responsible for a notable increase in the detection of Leishmaniinae infections in wild animals. Determining their infectiousness is of paramount importance in evaluating their epidemiological significance. One of the most efficient ways of determining infectiousness for vector borne diseases is xenodiagnosis with the appropriate vector. However, this is logistically very difficult to accomplish in the field, and an ideal solution is to find a molecular surrogate for xenodiagnosis. In this review we discuss different approaches to the problem by focusing on the infectiousness of Leishmania (Viannia) braziliensis in rodents under laboratory and field conditions. Comparisons with similar studies for other Leishmania species emphasizes that there are pivotal differences in the infectiousness and the importance of asymptomatic infections in different hosts. Potentially the most promising surrogate is the real time quantitative PCR (qPCR). However, its success depends on choosing a tissue that relates to the vector's feeding location and the parasite's tissue tropism. This requires detailed knowledge of the infection of each species in its wild hosts. We conclude that for L. (V.) braziliensis infections in wild rodents the tissue of choice for a molecular xenodiagnostic test, based on the qPCR is blood, providing that a significant number of samples must be examined.

Diversity and dissemination of viruses in pathogenic protozoa.

Viruses are the most abundant biological entities on Earth and play a significant role in the evolution of many organisms and ecosystems. In pathogenic protozoa, the presence of viruses has been linked to an increased risk of treatment failure and severe clinical outcome. Here, we studied the molecular epidemiology of the zoonotic disease cutaneous leishmaniasis in Peru and Bolivia through a joint evolutionary analysis of Leishmania braziliensis and their dsRNA Leishmania virus 1. We show that parasite populations circulate in tropical rainforests and are associated with single viral lineages that appear in low prevalence. In contrast, groups of hybrid parasites are geographically and ecologically more dispersed and associated with an increased prevalence, diversity and spread of viruses. Our results suggest that parasite gene flow and hybridization increased the frequency of parasite-virus symbioses, a process that may change the epidemiology of leishmaniasis in the region.

Leishmanicidal and healing effects of 3ß,6ß,16ß-trihydroxy lup-20 (29)-ene isolated from Combretum leprosum on Leishmania braziliensis infection in vitro and in vivo.

Treatment of cutaneous leishmaniasis depends on drugs that potentially cause serious side effects and resistance. Thus, topical therapies are attractive alternatives to the drugs currently used. 3ß, 6ß, 16ß-trihydroxylup-20 (29)-ene is a lupane triterpene isolated from Combretum leprosum Mart. leaves (CLF-1), with reports of in vitro antileishmanial effect against L. amazonensis and to promote lesion healing in animal model. Herein, we evaluated the in vitro and in vivo antileishmanial and healing effects of CLF-1 against L. braziliensis. CLF-1 treatment showed low toxicity in macrophages and significantly reduced parasite load in vitro. CLF-1 induced higher IL-12 and TNF-α production and more discrete IL-4 and IL-10 production. For in vivo evaluation, a CLF-1 cream formulation was prepared to treat hamsters infected with L. braziliensis. CLF-1 treatment was able to reduce parasite load of the infected skin and lymph node more efficiently than the conventional treatment. Histopathological analysis indicated a strong inflammatory response accompanied by an important healing response. Data from this study indicate that topical CLF-1 treatment was effective and non-toxic in L. braziliensis infected hamsters suggesting its potential for further development as a future therapeutic intervention.

Unveiling drug-tolerant and persister-like cells in Leishmania braziliensis lines derived from patients with cutaneous leishmaniasis.

Introduction: Resistance against anti-Leishmania drugs (DR) has been studied for years, giving important insights into long-term adaptations of these parasites to drugs, through genetic modifications. However, microorganisms can also survive lethal drug exposure by entering into temporary quiescence, a phenomenon called drug tolerance (DT), which is rather unexplored in Leishmania. Methods: We studied a panel of nine Leishmania braziliensis strains highly susceptible to potassium antimonyl tartrate (PAT), exposed promastigotes to lethal PAT pressure, and compared several cellular and molecular parameters distinguishing DT from DR. Results and discussion: We demonstrated in vitro that a variable proportion of cells remained viable, showing all the criteria of DT and not of DR: i) signatures of quiescence, under drug pressure: reduced proliferation and significant decrease of rDNA transcription; ii) reversibility of the phenotype: return to low IC50 after removal of drug pressure; and iii) absence of significant genetic differences between exposed and unexposed lineages of each strain and absence of reported markers of DR. We found different levels of quiescence and DT among the different L. braziliensis strains. We provide here a new in-vitro model of drug-induced quiescence and DT in Leishmania. Research should be extended in vivo, but the current model could be further exploited to support R&D, for instance, to guide the screening of compounds to overcome the quiescence resilience of the parasite, thereby improving the therapy of leishmaniasis.

Leishmania braziliensis exosomes activate human macrophages to produce proinflammatory mediators.

Exosomes, organelles measuring 30-200nm, are secreted by various cell types. Leishmania exosomes consist of many proteins, including heat shock proteins, annexins, Glycoprotein 63, proteins exerting signaling activity and those containing mRNA and miRNA. Studies have demonstrated that Leishmania donovani exosomes downregulate IFN-γ and inhibit the expression of microbicidal molecules, such as TNF and nitric oxide, thus creating a microenvironment favoring parasite proliferation. Despite lacking immunological memory, data in the literature suggest that, following initial stimulation, mononuclear phagocytes may become "trained" to respond more effectively to subsequent stimuli. Here we characterized the effects of macrophage sensitization using L. braziliensis exosomes prior to infection by the same pathogen. Human macrophages were stimulated with L. braziliensis exosomes and then infected with L. braziliensis. Higher levels of IL-1ß and IL-6 were detected in cultures sensitized prior to infection compared to unstimulated infected cells. Moreover, stimulation with L. braziliensis exosomes induced macrophage production of IL-1ß, IL-6, IL-10 and TNF. Inhibition of exosome secretion by L. braziliensis prior to macrophage infection reduced cytokine production and produced lower infection rates than untreated infected cells. Exosome stimulation also induced the consumption/regulation of NLRP3 inflammasome components in macrophages, while the blockade of NLRP3 resulted in lower levels of IL-6 and IL-1ß. Our results suggest that L. braziliensis exosomes stimulate macrophages, leading to an exacerbated inflammatory state that may be NLRP3-dependent.