CD4+ and CD8+ T-cell multi-epitope chimeric protein associated with an MPLA adjuvant induce protective efficacy and long-term immunological memory for the immunoprophylaxis of American Tegumentary Leishmaniasis.

American Tegumentary Leishmaniasis (ATL) is a disease of high severity and incidence in Brazil, in addition to being a worldwide concern in public health. Leishmania amazonensis is one of the etiological agents of ATL, and the inefficiency of control measures, associated with the high toxicity of the treatment and the lack of effective immunoprophylactic strategies, makes the development of vaccines indispensable and imminent. In this light, the present study proposes to elaborate a chimeric protein (rChiP), based on the fusion of multiple epitopes of CD4+/CD8+ T cells, identified in the immunoproteome of the parasites L. amazonensis and L. braziliensis. The designed chimeric protein was tested in the L. amazonensis murine model of infection using the following formulations: 25 µg of the rChiP in saline (rChiP group) and 25 µg of the rChiP plus 25 µg of MPLA-PHAD® (rChiP+MPLA group). After completing immunization, CD4+ and CD8+ T cells, stimulated with SLa-Antigen or rChiP, showed an increased production of nitric oxide and intracytoplasmic pro-inflammatory cytokines, in addition to the generation of central and effector memory T cells. rChiP and rChiP+MPLA formulations were able to promote an effective protection against L. amazonensis infection determined by a reduction in the development of skin lesions and lower parasitic burden. Reduction in the development of skin lesions and lower parasitic burden in the vaccinated groups were associated with an increase of nitrite, CD4+/CD8+IFN-γ+TNF-α+ and CD4+/CD8+CD44highCD62Lhigh/low T cells, IgGTotal, IgG2a, and lower rates of IgG1 and CD4+/CD8+IL-10+. This data suggests that proposed formulations could be considered potential tools to prevent ATL.

Association of Intestinal Helminthiasis with Disseminated Leishmaniasis, Brazil.

Disseminated leishmaniasis is an emerging clinical form of Leishmania braziliensis infection. Evidence shows that co-infection by L. braziliensis and intestinal helminths does not affect clinical manifestations or response to therapy in cutaneous leishmaniasis patients. We evaluated whether co-infection was associated with those aspects in disseminated leishmaniasis patients in Brazil.

Assessing proteases and enzymes of the trypanothione system in subpopulations of Leishmania (Viannia) braziliensis Thor strain during macrophage infection.

BACKGROUND: Leishmania (Viannia) braziliensis Thor strain exhibits a heterogeneous composition comprised of subpopulations with varying levels of infectivity. Clonal subpopulations were previously obtained from the strain Thor by sorting single-parasites and proceeding cultivation. The subpopulations used in this study are named Thor03, Thor 10 and Thor22. OBJECTIVES: Phenotypic characteristics of the parasite, specially focusing on virulence factors and resistance to the antimicrobial mechanisms of macrophages, were investigate in these subpopulations. METHODS: Cellular and molecular biology, as well as biochemistry approaches were applied to obtain the data analysed in this study. FINDINGS: Relative quantification of gene expression was measured for calpain, cysteine protease B (CPB), and subtilisin proteases but no significant differences in these genes' expression among subpopulations was observed. However, subtilisin and CPB proteins were assessed as more abundant in Thor03 by fluorescence-labelled flow cytometry technique. Western Blotting assays, as semi-quantitative analysis in gel, showed higher concentrations of subtilisin (110 to 50 kDa) and CPB (40 to 18 kDa) in extract of intracellular amastigotes from subpopulations Thor03 and Thor10 and calpain (60 to 25 kDa) showed no significant differences among subpopulations. Complementary, higher trypanothione reductase activity was observed in Thor10 intracellular amastigotes and assays of susceptibility to hydrogen peroxide-inducing agents and nitric oxide donors conducted with promastigotes revealed greater resistance to in vitro oxidative stress induction for Thor10, followed by Thor03. MAIN CONCLUSIONS: The data obtained for the virulence factors explored here suggest how multiple coexisting phenotypic-distinct subpopulations may contribute in adaptability of a single L. (V.) braziliensis strain during infection in the host cells.

Analysis of clinical cure outcome, macrophages number, cytokines levels and expression of annexin-A1 in the cutaneous infection in patients with Leishmania braziliensis.

BACKGROUND: Leishmania braziliensis, a protozoan prevalent in Brazil, is the known causative agent of cutaneous leishmaniasis (CL). The activation of M1 macrophages is a pivotal factor in the host's ability to eliminate the parasite, whereas M2 macrophages may facilitate parasite proliferation. This study analyzed the clinical outcomes of CL and the patients' immunological profiles, focusing on the prevalence of M1 and M2 macrophages, cytokine production, and annexin-A1 (ANXA1) expression in the lesion. METHODS: Data were obtained by polymerase chain reaction (PCR) and histopathological, immunofluorescence, and cytokine analyses. RESULTS: Patients with exudative and cellular reaction-type (ECR)-type lesions that healed within 90 days showed a significant increase in M1. Conversely, patients with ECR and exudative and granulomatous reaction (EGR)types, who healed within 180 days, showed an elevated number of M2. Cytokines interferon (IFN)-γ and tumor necrosis factor (TNF)-α were higher in ECR lesions that resolved within 90 days (P<0.05). In contrast, IL-9 and IL-10 levels significantly increased in both ECR and EGR lesions that healed after 180 days (P<0.001). The production of IL-21, IL-23 and TGF-ß was increased in patients with ECR or EGR lesions that healed after 180 days (P<0.05). The expression of ANXA1 was higher in M2 within ECR-type lesions in patients who healed after 180 days (P<0.05). CONCLUSIONS: These findings suggest that the infectious microenvironment induced by L. braziliensis affects the differentiation of M1 and M2 macrophages, cytokine release, and ANXA1 expression, thereby influencing the healing capacity of patients. Therefore, histopathological and immunological investigations may improve the selection of CL therapy.

Establishment of a laboratory colony of Pressatia choti (Diptera: Psychodidae), a suspected vector of Leishmania braziliensis.

BACKGROUND: Pressatia choti is a common sand fly found in the Atlantic Forest of Brazil, which is suspected to be involved in the transmission of Leishmania braziliensis. Herein, we aimed to establish a Pr. choti laboratory colony. METHODS: Wild-caught female sand flies were blood fed on hamsters and maintained under controlled conditions (temperature: 26 °C; relative humidity: 70%). RESULTS: Of the 301 collected female sandflies, 288 were identified as Pr. choti. The life cycle duration ranged from 31 to 56 days. CONCLUSIONS: We successfully established a Pr. choti colony, whose biological parameters were similar to those of other neotropical sand flies.

A Case of Cutaneous Leishmaniasis with Mucosal Involvement in the Northern United States.

BACKGROUND: Cutaneous leishmaniasis (CL) is a vector-borne parasitic infection endemic to many sub-tropical regions worldwide. In the Americas, Leishmania braziliensis is responsible for most reported CL cases. Variable symptom presentation and susceptibility to secondary infection make diagnosing CL a difficult proposition for physicians who may not encounter cases frequently. CASE REPORT: We present the case of a 50-year-old man with multiple progressive lesions, diagnosed initially as a bacterial infection, who presented to a North American emergency department after several unsuccessful trials of antibiotic therapy. Eventually, polymerase chain reaction testing of a wound biopsy sample confirmed the presence of L. braziliensis. After a complicated course, the patient's infection resolved after tailored antiparasitic therapy. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: This case highlights the need to include travel history in the evaluation of atypical dermatologic infections.

Transcriptomics analysis highlights potential ways in human pathogenesis in Leishmania braziliensis infected with the viral endosymbiont LRV1.

The parasite Leishmania (Viannia) braziliensis is widely distributed in Brazil and is one of the main species associated with human cases of different forms of tegumentary leishmaniasis (TL) such as cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML). The mechanisms underlying the pathogenesis of TL are still not fully understood, but it is known that factors related to the host and the parasite act in a synergistic and relevant way to direct the response to the infection. In the host, macrophages have a central connection with the parasite and play a fundamental role in the defense of the organism due to their ability to destroy intracellular parasites and present antigens. In the parasite, some intrinsic factors related to the species or even the strain analyzed are fundamental for the outcome of the disease. One of them is the presence of Leishmania RNA Virus 1 (LRV1), an endosymbiont virus that parasitizes some species of Leishmania that triggers a cascade of signals leading to a more severe TL phenotype, such as ML. One of the strategies for understanding factors associated with the immune response generated after Leishmania/host interaction is through the analysis of molecular patterns after infection. Thus, the gene expression profile in human monocyte-derived macrophages obtained from healthy donors infected in vitro with L. braziliensis positive (LbLRV1+) and negative (LbLRV1-) for LRV1 was evaluated. For this, the microarray assay was used and 162 differentially expressed genes were identified in the comparison LbLRV1+ vs. LbLRV1-, 126 upregulated genes for the type I and II interferons (IFN) signaling pathway, oligoadenylate synthase OAS/RNAse L, non-genomic actions of vitamin D3 and RIG-I type receptors, and 36 down-regulated. The top 10 downregulated genes along with the top 10 upregulated genes were considered for analysis. Type I interferon (IFNI)- and OAS-related pathways results were validated by RT-qPCR and Th1/Th2/Th17 cytokines were analyzed by Cytometric Bead Array (CBA) and enzyme-linked immunosorbent assay (ELISA). The microarray results validated by RT-qPCR showed differential expression of genes related to IFNI-mediated pathways with overexpression of different genes in cells infected with LbLRV1+ compared to LbLRV1- and to the control. No significant differences were found in cytokine levels between LbLRV1+ vs. LbLRV1- and control. The data suggest the activation of gene signaling pathways associated with the presence of LRV1 has not yet been reported so far. This study demonstrates, for the first time, the activation of the OAS/RNase L signaling pathway and the non-genomic actions of vitamin D3 when comparing infections with LbLRV1+ versus LbLRV1- and the control. This finding emphasizes the role of LRV1 in directing the host's immune response after infection, underlining the importance of identifying LRV1 in patients with TL to assess disease progression.

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.

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.

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.

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.

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.

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.

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.

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.