Pharmacokinetics, Dose-Proportionality, and Tolerability of Intravenous Tanespimycin (17-AAG) in Single and Multiple Doses in Dogs: A Potential Novel Treatment for Canine Visceral Leishmaniasis.

In the New World, dogs are considered the main reservoir of visceral leishmaniasis (VL). Due to inefficacies in existing treatments and the lack of an efficient vaccine, dog culling is one of the main strategies used to control disease, making the development of new therapeutic interventions mandatory. We previously showed that Tanespimycin (17-AAG), a Hsp90 inhibitor, demonstrated potential for use in leishmaniasis treatment. The present study aimed to test the safety of 17-AAG in dogs by evaluating plasma pharmacokinetics, dose-proportionality, and the tolerability of 17-AAG in response to a dose-escalation protocol and multiple administrations at a single dose in healthy dogs. Two protocols were used: Study A: four dogs received variable intravenous (IV) doses (50, 100, 150, 200, or 250 mg/m2) of 17-AAG or a placebo (n = 4/dose level), using a cross-over design with a 7-day "wash-out" period; Study B: nine dogs received three IV doses of 150 mg/m2 of 17-AAG administered at 48 h intervals. 17-AAG concentrations were determined by a validated high-performance liquid chromatographic (HPLC) method: linearity (R2 = 0.9964), intra-day precision with a coefficient of variation (CV) ≤ 8%, inter-day precision (CV ≤ 20%), and detection and quantification limits of 12.5 and 25 ng/mL, respectively. In Study A, 17-AAG was generally well tolerated. However, increased levels of liver enzymes-alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase (GGT)-and bloody diarrhea were observed in all four dogs receiving the highest dosage of 250 mg/m2. After single doses of 17-AAG (50-250 mg/m2), maximum plasma concentrations (Cmax) ranged between 1405 ± 686 and 9439 ± 991 ng/mL, and the area under the curve (AUC) plotting plasma concentration against time ranged between 1483 ± 694 and 11,902 ± 1962 AUC 0-8 h µg/mL × h, respectively. Cmax and AUC parameters were dose-proportionate between the 50 and 200 mg/m2 doses. Regarding Study B, 17-AAG was found to be well tolerated at multiple doses of 150 mg/m2. Increased levels of liver enzymes-ALT (28.57 ± 4.29 to 173.33 ± 49.56 U/L), AST (27.85 ± 3.80 to 248.20 ± 85.80 U/L), and GGT (1.60 ± 0.06 to 12.70 ± 0.50 U/L)-and bloody diarrhea were observed in only 3/9 of these dogs. After the administration of multiple doses, Cmax and AUC 0-48 h were 5254 ± 2784 µg/mL and 6850 ± 469 µg/mL × h in plasma and 736 ± 294 µg/mL and 7382 ± 1357 µg/mL × h in tissue transudate, respectively. In conclusion, our results demonstrate the potential of 17-AAG in the treatment of CVL, using a regimen of three doses at 150 mg/m2, since it presents the maintenance of high concentrations in subcutaneous interstitial fluid, low toxicity, and reversible hepatotoxicity.

Elucidating the role played by bone marrow in visceral leishmaniasis.

Leishmaniasis is a widespread group of infectious diseases that significantly impact global health. Despite high prevalence, leishmaniasis often receives inadequate attention in the prioritization of measures targeting tropical diseases. The causative agents of leishmaniasis are protozoan parasites of the Leishmania genus, which give rise to a diverse range of clinical manifestations, including cutaneous and visceral forms. Visceral leishmaniasis (VL), the most severe form, can be life-threatening if left untreated. Parasites can spread systemically within the body, infecting a range of organs, such as the liver, spleen, bone marrow and lymph nodes. Natural reservoirs for these protozoa include rodents, dogs, foxes, jackals, and wolves, with dogs serving as the primary urban reservoir for Leishmania infantum. Dogs exhibit clinical and pathological similarities to human VL and are valuable models for studying disease progression. Both human and canine VL provoke clinical symptoms, such as organ enlargement, fever, weight loss and abnormal gamma globulin levels. Hematologic abnormalities have also been observed, including anemia, leukopenia with lymphocytosis, neutropenia, and thrombocytopenia. Studies in dogs have linked these hematologic changes in peripheral blood to alterations in the bone marrow. Mouse models of VL have also contributed significantly to our understanding of the mechanisms underlying these hematologic and bone marrow abnormalities. This review consolidates information on hematological and immunological changes in the bone marrow of humans, dogs, and mice infected with Leishmania species causing VL. It includes findings on the role of bone marrow as a source of parasite persistence in internal organs and VL development. Highlighting gaps in current knowledge, the review emphasizes the need for future research to enhance our understanding of VL and identify potential targets for novel diagnostic and therapeutic approaches.

Leishmania infection alters macrophage and dendritic cell migration in a three-dimensional environment.

Background: Leishmaniasis results in a wide spectrum of clinical manifestations, ranging from skin lesions at the site of infection to disseminated lesions in internal organs, such as the spleen and liver. While the ability of Leishmania-infected host cells to migrate may be important to lesion distribution and parasite dissemination, the underlying mechanisms and the accompanying role of host cells remain poorly understood. Previously published work has shown that Leishmania infection inhibits macrophage migration in a 2-dimensional (2D) environment by altering actin dynamics and impairing the expression of proteins involved in plasma membrane-extracellular matrix interactions. Although it was shown that L. infantum induces the 2D migration of dendritic cells, in vivo cell migration primarily occurs in 3-dimensional (3D) environments. The present study aimed to investigate the migration of macrophages and dendritic cells infected by Leishmania using a 3-dimensional environment, as well as shed light on the mechanisms involved in this process. Methods: Following the infection of murine bone marrow-derived macrophages (BMDM), human macrophages and human dendritic cells by L. amazonensis, L. braziliensis, or L. infantum, cellular migration, the formation of adhesion complexes and actin polymerization were evaluated. Results: Our results indicate that Leishmania infection inhibited 3D migration in both BMDM and human macrophages. Reduced expression of proteins involved in adhesion complex formation and alterations in actin dynamics were also observed in Leishmania-infected macrophages. By contrast, increased human dendritic cell migration in a 3D environment was found to be associated with enhanced adhesion complex formation and increased actin dynamics. Conclusion: Taken together, our results show that Leishmania infection inhibits macrophage 3D migration, while enhancing dendritic 3D migration by altering actin dynamics and the expression of proteins involved in plasma membrane extracellular matrix interactions, suggesting a potential association between dendritic cells and disease visceralization.

Leishmania amazonensis infection impairs VLA-4 clustering and adhesion complex assembly at the adhesion site of J774 cells.

Cutaneous leishmaniasis is an infectious disease that may lead to a single or multiple disseminated cutaneous lesions. The mechanisms involved in Leishmania dissemination to different areas of the skin and the internal organs remain poorly understood. Evidence shows that Very Late Antigen-4 (VLA-4)-dependent phagocyte adhesion is impaired by Leishmania infection, which may be related to the mechanisms of parasite dissemination. We investigated factors potentially associated with decreased VLA-4-mediated adhesion in Leishmania-infected macrophages, including lipid raft-mediated VLA-4 mobilization along the cellular membrane, integrin cluster formation at the cell base (adhesion site), and focal adhesion complex assembly. Phagocytes treated with Methyl-ß-Cyclodextrin (MßCD) demonstrated reduced adhesion, similarly to Leishmania amazonensis-infected J774 cells. Infected and MßCD-treated macrophages presented decreased VLA-4 mobilization to the adhesion plane, as well as reduced integrin clustering. Leishmania amazonensis-infected cells exhibited talin depletion, as well as a decreased mobilization of adhesion complex proteins, such as talin and viculin, which were associated with lower VLA-4 concentrations at the adhesion site and limited cell-spreading. Our results suggest that Leishmania infection may modulate the firm adhesion phase of the cell-spreading process, which could contribute to the bloodstream dissemination of infected cells.

Autophagy in protists and their hosts: When, how and why?

Pathogenic protists are a group of organisms responsible for causing a variety of human diseases including malaria, sleeping sickness, Chagas disease, leishmaniasis, and toxoplasmosis, among others. These diseases, which affect more than one billion people globally, mainly the poorest populations, are characterized by severe chronic stages and the lack of effective antiparasitic treatment. Parasitic protists display complex life-cycles and go through different cellular transformations in order to adapt to the different hosts they live in. Autophagy, a highly conserved cellular degradation process, has emerged as a key mechanism required for these differentiation processes, as well as other functions that are crucial to parasite fitness. In contrast to yeasts and mammals, protist autophagy is characterized by a modest number of conserved autophagy-related proteins (ATGs) that, even though, can drive the autophagosome formation and degradation. In addition, during their intracellular cycle, the interaction of these pathogens with the host autophagy system plays a crucial role resulting in a beneficial or harmful effect that is important for the outcome of the infection. In this review, we summarize the current state of knowledge on autophagy and other related mechanisms in pathogenic protists and their hosts. We sought to emphasize when, how, and why this process takes place, and the effects it may have on the parasitic cycle. A better understanding of the significance of autophagy for the protist life-cycle will potentially be helpful to design novel anti-parasitic strategies.

Overall Survival and Response with Nivolumab and Relatlimab in Advanced Melanoma.

BACKGROUND: A phase 2/3 trial ­ A Study of Relatlimab Plus Nivolumab Versus Nivolumab Alone in Participants With Advanced Melanoma (RELATIVITY-047) ­ evaluated nivolumab + relatlimab as a fixed-dose combination and found a significant progression-free survival (PFS) benefit over nivolumab monotherapy in previously untreated unresectable or metastatic melanoma. We now report updated PFS and safety data and the first results for overall survival (OS) and objective response rate (ORR). METHODS: Patients were randomly assigned 1:1 to receive nivolumab 480 mg and relatlimab 160 mg fixed-dose combination or nivolumab 480 mg alone, given intravenously every 4 weeks. PFS (primary end point) according to the Response Evaluation Criteria in Solid Tumors, version 1.1, was assessed by blinded independent central review (BICR). Secondary end points, tested hierarchically, were OS and then ORR per Response Evaluation Criteria in Solid Tumors, version 1.1, per BICR. RESULTS: At a median follow-up of 19.3 months, median PFS according to BICR was 10.2 months (95% confidence interval [CI], 6.5 to 14.8) with nivolumab + relatlimab versus 4.6 months (95% CI, 3.5 to 6.4) with nivolumab (hazard ratio, 0.78; 95% CI, 0.64 to 0.94). Median OS was not reached (NR) (95% CI, 34.2 to NR) with nivolumab + relatlimab versus 34.1 months (95% CI, 25.2 to NR) with nivolumab (hazard ratio, 0.80; 95% CI, 0.64 to 1.01; P=0.059) (prespecified value for statistical significance, P≤0.043). ORRs per BICR were 43.1% (95% CI, 37.9 to 48.4) versus 32.6% (95% CI, 27.8 to 37.7), respectively. Grade 3/4 treatment-related adverse events were observed in 21.1% of patients treated with nivolumab + relatlimab versus 11.1% treated with nivolumab. CONCLUSIONS: The fixed-dose combination of nivolumab + relatlimab showed consistent PFS benefit versus nivolumab with approximately 6 months of additional median follow-up. The combination treatment did not reach the preplanned statistical threshold for OS, with a 10.3 percentage-point difference in ORR. Grade 3/4 treatment-related adverse events were more frequent with nivolumab + relatlimab versus nivolumab. (Funded by Bristol Myers Squibb; ClinicalTrials.gov number, NCT03470922.)

Leishmania infantum Defective in Lipophosphoglycan Biosynthesis Interferes With Activation of Human Neutrophils.

Visceral leishmaniasis (VL) is often associated with hematologic manifestations that may interfere with neutrophil response. Lipophosphoglycan (LPG) is a major molecule on the surface of Leishmania promastigotes, which has been associated with several aspects of the parasite-vector-host interplay. Here, we investigated how LPG from Leishmania (L.) infantum, the principal etiological agent of VL in the New World, influences the initial establishment of infection during interaction with human neutrophils in an experimental setting in vitro. Human neutrophils obtained from peripheral blood samples were infected with either the wild-type L. infantum (WT) strain or LPG-deficient mutant (∆lpg1). In this setting, ∆lpg1 parasites displayed reduced viability compared to WT L. infantum; such finding was reverted in the complemented ∆lpg1+LPG1 parasites at 3- and 6-h post-infection. Confocal microscopy experiments indicated that this decreased survival was related to enhanced lysosomal fusion. In fact, LPG-deficient L. infantum parasites more frequently died inside neutrophil acidic compartments, a phenomenon that was reverted when host cells were treated with Wortmannin. We also observed an increase in the secretion of the neutrophil collagenase matrix metalloproteinase-8 (MMP-8) by cells infected with ∆lpg1 L. infantum compared to those that were infected with WT parasites. Furthermore, collagen I matrix degradation was found to be significantly increased in ∆lpg1 parasite-infected cells but not in WT-infected controls. Flow cytometry analysis revealed a substantial boost in production of reactive oxygen species (ROS) during infection with either WT or ∆lpg1 L. infantum. In addition, killing of ∆lpg1 parasites was shown to be more dependent on the ROS production than that of WT L. infantum. Notably, inhibition of the oxidative stress with Apocynin potentially fueled ∆lpg1 L. infantum fitness as it increased the intracellular parasite viability. Thus, our observations demonstrate that LPG may be a critical molecule fostering parasite survival in human neutrophils through a mechanism that involves cellular activation and generation of free radicals.

In Vitro Differentiation of Human Dendritic Cells and their Markers in Leishmania Infection.

Leishmaniasis comprises a collection of clinical manifestations associated with the infection of obligate intracellular protozoans, Leishmania. The life cycle of Leishmania parasites consists of two alternating life stages (amastigotes and promastigotes), during which parasites reside within either arthropod vectors or vertebrate hosts, respectively. Notably, the complex interactions between Leishmania parasites and several cells of the immune system largely influence the outcome of infection. Importantly, although macrophages are known to be the main host niche for Leishmania replication, parasites are also phagocytosed by other innate immune cells, such as neutrophils and dendritic cells (DCs). DCs play a major role in bridging the innate and adaptive branches of immunity and thus orchestrate immune responses against a wide range of pathogens. The mechanisms by which Leishmania and DCs interact remain unclear and involve aspects of pathogen capture, the dynamics of DC maturation and activation, DC migration to draining lymph node (dLNs), and antigen presentation to T cells. Although a large body of studies support the notion that DCs play a dual role in modulating immune responses against Leishmania, the participation of these cells in susceptibility or resistance to Leishmania remains poorly understood. After infection, DCs undergo a maturation process associated with the upregulation of surface major histocompatibility complex (MHC) II, in addition to costimulatory molecules (namely, CD40, CD80, and CD86). Understanding the role of DCs in infection outcome is crucial to developing therapeutic and prophylactic strategies to modulate the immune response against Leishmania. This paper describes a method for the characterization of Leishmania-DC interaction. This detailed protocol provides guidance throughout the steps of DC differentiation, the characterization of cell surface molecules, and infection protocols, allowing scientists to investigate DC response to Leishmania infection and gain insight into the roles played by these cells in the course of infection.

Relatlimab and Nivolumab versus Nivolumab in Untreated Advanced Melanoma.

BACKGROUND: Lymphocyte-activation gene 3 (LAG-3) and programmed death 1 (PD-1) are distinct inhibitory immune checkpoints that contribute to T-cell exhaustion. The combination of relatlimab, a LAG-3-blocking antibody, and nivolumab, a PD-1-blocking antibody, has been shown to be safe and to have antitumor activity in patients with previously treated melanoma, but the safety and activity in patients with previously untreated melanoma need investigation. METHODS: In this phase 2-3, global, double-blind, randomized trial, we evaluated relatlimab and nivolumab as a fixed-dose combination as compared with nivolumab alone when administered intravenously every 4 weeks to patients with previously untreated metastatic or unresectable melanoma. The primary end point was progression-free survival as assessed by blinded independent central review. RESULTS: The median progression-free survival was 10.1 months (95% confidence interval [CI], 6.4 to 15.7) with relatlimab-nivolumab as compared with 4.6 months (95% CI, 3.4 to 5.6) with nivolumab (hazard ratio for progression or death, 0.75 [95% CI, 0.62 to 0.92]; P = 0.006 by the log-rank test). Progression-free survival at 12 months was 47.7% (95% CI, 41.8 to 53.2) with relatlimab-nivolumab as compared with 36.0% (95% CI, 30.5 to 41.6) with nivolumab. Progression-free survival across key subgroups favored relatlimab-nivolumab over nivolumab. Grade 3 or 4 treatment-related adverse events occurred in 18.9% of patients in the relatlimab-nivolumab group and in 9.7% of patients in the nivolumab group. CONCLUSIONS: The inhibition of two immune checkpoints, LAG-3 and PD-1, provided a greater benefit with regard to progression-free survival than inhibition of PD-1 alone in patients with previously untreated metastatic or unresectable melanoma. Relatlimab and nivolumab in combination showed no new safety signals. (Funded by Bristol Myers Squibb; RELATIVITY-047 ClinicalTrials.gov number, NCT03470922.).

Abiraterone and Olaparib for Metastatic Castration-Resistant Prostate Cancer.

BACKGROUND: Preclinical studies and results of a phase 2 trial of abiraterone and olaparib suggest a combined antitumor effect when the poly(adenosine diphosphate[ADP]-ribose) polymerase inhibitor olaparib is combined with next-generation hormonal agent abiraterone to treat metastatic castration-resistant prostate cancer (mCRPC). METHODS: We conducted a double-blind, phase 3 trial of abiraterone and olaparib versus abiraterone and placebo in patients with mCRPC in the first-line setting. Patients were enrolled regardless of homologous recombination repair gene mutation (HRRm) status. HRRm status was determined following enrollment by tumor tissue and circulating tumor DNA tests. Patients were randomly assigned (1:1) to receive abiraterone (1000 mg once daily) plus prednisone or prednisolone with either olaparib (300 mg twice daily) or placebo. The primary end point was imaging-based progression-free survival (ibPFS) by investigator assessment. Overall survival was among the secondary end points. RESULTS: At this planned primary analysis at the first data cutoff, median ibPFS was significantly longer in the abiraterone and olaparib arm than in the abiraterone and placebo arm (24.8 vs. 16.6 months; hazard ratio, 0.66; 95% confidence interval [CI], 0.54 to 0.81; P<0.001) and was consistent with blinded independent central review (hazard ratio, 0.61; 95% CI, 0.49 to 0.74). At this data cutoff, overall survival data were immature (28.6% maturity; hazard ratio, 0.86; 95% CI, 0.66 to 1.12; P=0.29). The safety profile of olaparib and abiraterone was consistent with the known safety profiles of the individual drugs. The most common adverse events in the abiraterone and olaparib arm were anemia, fatigue/asthenia, and nausea. CONCLUSIONS: At primary analysis at this first data cutoff, abiraterone combined with olaparib significantly prolonged ibPFS compared with abiraterone and placebo as first-line treatment for patients with mCRPC enrolled irrespective of HRRm status. (Funded by AstraZeneca and Merck Sharp & Dohme, LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA; ClinicalTrials.gov number, NCT03732820.)

Investigating the Phagocytosis of Leishmania using Confocal Microscopy.

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up Leishmania parasites by phagocytosis, consisting of recognizing ligands on parasite surfaces by multiple host cell receptors. Binding of Leishmania to macrophage membranes occurs through complement receptor type 1 (CR1) and complement receptor type 3 (CR3) and Pattern Recognition Receptors. Lipophosphoglycan (LPG) and 63 kDa glycoprotein (gp63) are the main ligands involved in macrophage-Leishmania interactions. Following the initial recognition of parasite ligands by host cell receptors, parasites become internalized, survive, and multiply within parasitophorous vacuoles. The maturation process of Leishmania-induced vacuoles involves the acquisition of molecules from intracellular vesicles, including monomeric G protein Rab 5 and Rab 7, lysosomal associated membrane protein 1 (LAMP-1), lysosomal associated membrane protein 2 (LAMP-2), and microtubule-associated protein 1A/1B-light chain 3 (LC3). Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells using confocal microscopy, including (i) binding (ii) internalization, and (iii) phagosome maturation. By adding to the body of knowledge surrounding these determinants of infection outcome, we hope to improve the understanding of the pathogenesis of Leishmania infection and support the eventual search for novel chemotherapeutic targets.

Obtainment of Macrophages from Human Monocytes to Assess Leishmania braziliensis Infection Rate and Innate Host Immune Response.

Macrophages are multifunctional cells essential to the immune system function, and the primary host cell in Leishmania braziliensis (Lb) infection. These cells are specialized in microorganism recognition and phagocytosis, but also activate other immune cells and present antigens, as well as promote inflammation and tissue repair. Here, we describe a protocol to obtain mononuclear cells from peripheral blood (PBMC) of healthy donors to separate monocytes that then differentiate into macrophages. These cells can then be infected in vitro at different Lb concentrations to evaluate the ability to control infection, as well as evaluate host cell immune response, which can be measured by several methods. PBMCs were first isolated by centrifuging with Ficoll-Hypaque gradient and then plated to allow monocytes to adhere to culture plates; non-adherent cells were removed by washing. Next, adherent cells were cultured with macrophage-colony stimulating factor (M-CSF) for 7 days to induce macrophage differentiation. We suggest plating 2 x 106 cells per well on 24-well plates in order to obtain 2 x 105 macrophages. Fully differentiated macrophages can then be infected with Lb for 4 or 24 hours. This protocol results in a significant percentage of infected cells, which can be assessed by optical or fluorescence microscopy. In addition to infection index, parasite load can be measured by counting the numbers of parasites inside each cell. Further molecular and functional assays can also be performed in culture supernatants or within the macrophages themselves, which allows this protocol to be applied in a variety of contexts and also adapted to other intracellular parasite species.

Cell Migration and Cell Adhesion Assays to Investigate Leishmania-Host Cell Interaction.

Leishmania is an intracellular protozoan parasite that causes a broad spectrum of clinical manifestations, ranging from self-resolving localized cutaneous lesions to a highly fatal visceral form of the disease. An estimated 12 million people worldwide are currently infected, and another 350 million face risk of infection. It is known that host cells infected by Leishmania parasites, such as macrophages or dendritic cells, can migrate to different host tissues, yet how migration contributes to parasite dissemination and homing remains poorly understood. Therefore, assessing these parasites' ability to modulate host cell response, adhesion, and migration will shed light on mechanisms involved in disease dissemination and visceralization. Cellular migration is a complex process in which cells undergo polarization and protrusion, allowing them to migrate. This process, regulated by actin and tubulin-based microtubule dynamics, involves different factors, including the modulation of cellular adhesion to the substrate. Cellular adhesion and migration processes have been investigated using several models. Here, we describe a method to characterize the migratory aspects of host cells during Leishmania infection. This detailed protocol presents the differentiation and infection of dendritic cells, the analysis of host cell motility and migration, and the formation of adhesion complexes and actin dynamics. This in vitro protocol aims to further elucidate mechanisms involved in Leishmania dissemination within vertebrate host tissues and can also be modified and applied to other cell migration studies.

Leishmania-Induced Dendritic Cell Migration and Its Potential Contribution to Parasite Dissemination.

Leishmania, an intracellular parasite species, causes lesions on the skin and in the mucosa and internal organs. The dissemination of infected host cells containing Leishmania is crucial to parasite survival and the establishment of infection. Migratory phenomena and the mechanisms underlying the dissemination of Leishmania-infected human dendritic cells (hDCs) remain poorly understood. The present study aimed to investigate differences among factors involved in hDC migration by comparing infection with visceral leishmaniasis (VL) induced by Leishmaniainfantum with diverse clinical forms of tegumentary leishmaniasis (TL) induced by Leishmaniabraziliensis or Leishmania amazonensis. Following the infection of hDCs by isolates obtained from patients with different clinical forms of Leishmania, the formation of adhesion complexes, actin polymerization, and CCR7 expression were evaluated. We observed increased hDC migration following infection with isolates of L. infantum (VL), as well as disseminated (DL) and diffuse (DCL) forms of cutaneous leishmaniasis (CL) caused by L. braziliensis and L. amazonensis, respectively. Increased expression of proteins involved in adhesion complex formation and actin polymerization, as well as higher CCR7 expression, were seen in hDCs infected with L. infantum, DL and DCL isolates. Together, our results suggest that hDCs play an important role in the dissemination of Leishmania parasites in the vertebrate host.

17-AAG-Induced Activation of the Autophagic Pathway in Leishmania Is Associated with Parasite Death.

The heat shock protein 90 (Hsp90) is thought to be an excellent drug target against parasitic diseases. The leishmanicidal effect of an Hsp90 inhibitor, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), was previously demonstrated in both in vitro and in vivo models of cutaneous leishmaniasis. Parasite death was shown to occur in association with severe ultrastructural alterations in Leishmania, suggestive of autophagic activation. We hypothesized that 17-AAG treatment results in the abnormal activation of the autophagic pathway, leading to parasite death. To elucidate this process, experiments were performed using transgenic parasites with GFP-ATG8-labelled autophagosomes. Mutant parasites treated with 17-AAG exhibited autophagosomes that did not entrap cargo, such as glycosomes, or fuse with lysosomes. ATG5-knockout (Δatg5) parasites, which are incapable of forming autophagosomes, demonstrated lower sensitivity to 17-AAG-induced cell death when compared to wild-type (WT) Leishmania, further supporting the role of autophagy in 17-AAG-induced cell death. In addition, Hsp90 inhibition resulted in greater accumulation of ubiquitylated proteins in both WT- and Δatg5-treated parasites compared to controls, in the absence of proteasome overload. In conjunction with previously described ultrastructural alterations, herein we present evidence that treatment with 17-AAG causes abnormal activation of the autophagic pathway, resulting in the formation of immature autophagosomes and, consequently, incidental parasite death.

Deciphering the Role Played by Autophagy in Leishmania Infection.

In recent decades, studies have shown that, depending on parasite species and host background, autophagy can either favor infection or promote parasite clearance. To date, relatively few studies have attempted to assess the role played by autophagy in Leishmania infection. While it has been consistently shown that Leishmania spp. induce autophagy in a variety of cell types, published results regarding the effects of autophagic modulation on Leishmania survival are contradictory. The present review, after a short overview of the general aspects of autophagy, aims to summarize the current body of knowledge surrounding how Leishmania spp. adaptively interact with macrophages, the host cells mainly involved in controlling leishmaniasis. We then explore the scarce studies that have investigated interactions between these parasite species and the autophagic pathway, and finally present a critical perspective on how autophagy influences infection outcome.

Proteomic Analysis Reveals a Predominant NFE2L2 (NRF2) Signature in Canonical Pathway and Upstream Regulator Analysis of Leishmania-Infected Macrophages.

CBA mice macrophages (MØ) control infection by Leishmania major and are susceptive to Leishmania amazonensis, suggesting that both parasite species induce distinct responses that play important roles in infection outcome. To evaluate the MØ responses to infection arising from these two Leishmania species, a proteomic study using a Multidimensional Protein Identification Technology (MudPIT) approach with liquid chromatography tandem mass spectrometry (LC-MS/MS) was carried out on CBA mice bone-marrow MØ (BMMØ). Following SEQUEST analysis, which revealed 2,838 proteins detected in BMMØ, data mining approach found six proteins significantly associated with the tested conditions. To investigate their biological significance, enrichment analysis was performed using Ingenuity Pathway Analysis (IPA). A three steps IPA approach revealed 4 Canonical Pathways (CP) and 7 Upstream Transcriptional Factors (UTFs) strongly associated with the infection process. NRF2 signatures were present in both CPs and UTFs pathways. Proteins involved in iron metabolism, such as heme oxigenase 1 (HO-1) and ferritin besides sequestosome (SQSMT1 or p62) were found in the NRF2 CPs and the NRF2 UTFs. Differences in the involvement of iron metabolism pathway in Leishmania infection was revealed by the presence of HO-1 and ferritin. Noteworty, HO-1 was strongly associated with L. amazonensis infection, while ferritin was regulated by both species. As expected, higher HO-1 and p62 expressions were validated in L. amazonensis-infected BMMØ, in addition to decreased expression of ferritin and nitric oxide production. Moreover, BMMØ incubated with L. amazonensis LPG also expressed higher levels of HO-1 in comparison to those stimulated with L. major LPG. In addition, L. amazonensis-induced uptake of holoTf was higher than that induced by L. major in BMMØ, and holoTf was also detected at higher levels in vacuoles induced by L. amazonensis. Taken together, these findings indicate that NRF2 pathway activation and increased HO-1 production, together with higher levels of holoTf uptake, may promote permissiveness to L. amazonensis infection. In this context, differences in protein signatures triggered in the host by L. amazonensis and L. major infection could drive the outcomes in distinct clinical forms of leishmaniasis.