New Immunological Markers in Chromoblastomycosis-The Importance of PD-1 and PD-L1 Molecules in Human Infection.

The pathogenesis of chromoblastomycosis (CBM) is associated with Th2 and/or T regulatory immune responses, while resistance is associated with a Th1 response. However, even in the presence of IFN-γ, fungi persist in the lesions, and the reason for this persistence is unknown. To clarify the factors associated with pathogenesis, this study aimed to determine the polarization of the cellular immune response and the densities of cells that express markers of exhaustion in the skin of CBM patients. In the skin of patients with CBM, a moderate inflammatory infiltrate was observed, characterized primarily by the occurrence of histiocytes. Analysis of fungal density allowed us to divide patients into groups that exhibited low and high fungal densities; however, the intensity of the inflammatory response was not related to mycotic loads. Furthermore, patients with CBM exhibited a significant increase in the number of CD4+ and CD8+ cells associated with a high density of IL-10-, IL-17-, and IFN-γ-producing cells, indicating the presence of a chronic and mixed cellular immune response, which was also independent of fungal load. A significant increase in the number of PD-1+ and PD-L1+ cells was observed, which may be associated with the maintenance of the fungus in the skin and the progression of the disease.

Prevalence and incidence of canine visceral leishmaniasis and its clinical-immunological features in an endemic area of the Brazilian Amazon.

BACKGROUND: A cohort study for 2 years period analysed the prevalence, incidence and clinical-immunological features of canine Leishmania (L.) chagasi-infection in 316 mongrel dogs in a visceral leishmaniasis-endemic area in Pará State, Brazil. OBJECTIVE/METHODS: Diagnosis of infection was performed by the indirect fluorescent antibody test (IFAT-IgG), the leishmanin skin test (LST) and a parasite search (from the popliteal lymph node aspiration) at the beginning of the study and at 6, 12 and 24 months intervals. RESULTS: IFAT/LST revealed three immune profiles of infection: (I) IFAT(+) /LST(-) (81), (II) IFAT(-) /LST(+) (17) and (III) IFAT(+) /LST(+) (13). Prevalence of profiles I, II and III were 25.6, 5.4 and 4.1%, and an overall prevalence 35.1%. Incidence of profiles I, II and III were 5.4, 0.3 and 0.0%, and an overall incidence 5.7% dogs per month. Incidence at the age ranges <1 year, ≥1 year, <7 years and ≥7 years evidenced a highest rate in the age range <1 year (6.6% dogs per month). Parasitological diagnosis was positive in 19% dogs at the prevalence (85.7% profile I), and in 11% at the incidence (100% profile I). The clinical picture of 179 infected dogs showed 145 (81%) of profile I (82% subclinical); 21 (11.7%) of profile II (100% subclinical); and 13 (7.3%) of profile III (84.6% subclinical). Conversion from subclinical to sick dogs was higher (p < 0.05) in profile I (40.2%) than in profiles II (5.8%) and III (9%). Immunological conversion showed that only 3.2% of profile I dogs (prevalence) converted to LST(+) (two at the end of the first 6 months and 1 after 24 months), while 82.3% of profile II dogs converted to IFAT(+) (11 in the first 6 months, whereas three after 12 months). A 100% death rate was observed in dogs from profile I alone. CONCLUSION: These results reinforce the need of adopting preventive strategies against CVL as early as in the first semester of the dog's life.

Gene Signatures of Symptomatic and Asymptomatic Clinical-Immunological Profiles of Human Infection by Leishmania (L.) chagasi in Amazonian Brazil.

Individuals infected with Leishmania (L.) chagasi may present different asymptomatic and symptomatic stages of infection, which vary in the clinical-immunological profiles that can be classified as asymptomatic infection (AI), subclinical resistant infection (SRI), indeterminate initial infection (III), subclinical oligosymptomatic infection (SOI), and symptomatic infection (SI) (=American visceral leishmaniasis, AVL). However, little is known about the molecular differences between individuals having each profile. Here, we performed whole-blood transcriptomic analyses of 56 infected individuals from Pará State (Brazilian Amazon), covering all five profiles. We then identified the gene signatures of each profile by comparing their transcriptome with those of 11 healthy individuals from the same area. Symptomatic individuals with SI (=AVL) and SOI profiles showed higher transcriptome perturbation when compared to those asymptomatic III, AI and SRI profiles, suggesting that disease severity may be associated with greater transcriptomic changes. Although the expression of many genes was altered on each profile, very few genes were shared among the profiles. This indicated that each profile has a unique gene signature. The innate immune system pathway was strongly activated only in asymptomatic AI and SRI profiles, suggesting the control of infection. In turn, pathways such as MHC Class II antigen presentation and NF-kB activation in B cells seemed to be specifically induced in symptomatic SI (=AVL) and SOI profiles. Moreover, cellular response to starvation was down-regulated in those symptomatic profiles. Overall, this study revealed five distinct transcriptional patterns associated to the clinical-immunological (symptomatic and asymptomatic) profiles of human L. (L.) chagasi-infection in the Brazilian Amazon.

Role of antigen-presenting cells in non-ulcerated skin lesions caused by Leishmania (Leishmania) infantum chagasi.

In Central America, infection by Leishmania (Leishmania) infantum chagasi causes visceral leishmaniasis and non-ulcerated cutaneous leishmaniasis (NUCL). This work aimed to evaluate the participation of subpopulations of antigen-presenting cells in skin lesions of patients affected by NUCL through double-staining immunohistochemistry using cellular and intracellular markers. Twenty-three skin biopsies from patients affected by NUCL were used. Histological sections stained by HE were used for histopathological study. Immunohistochemical studies were performed using primary antibodies against Langerhans cells, dermal dendritic cells, T lymphocytes, and the cytokines IL-12, IFN-γ, TNF-α, iNOS, and IL-10. The histopathological lesions were characterized by an inflammatory infiltrate, predominantly lymphohistiocytic, of variable intensity, with a diffuse arrangement associated with epithelioid granulomas and discreet parasitism. Double-staining immunohistochemistry showed higher participation of dendritic cells producing the proinflammatory cytokine IL-12 in relation to the other evaluated cytokines. Activation of the cellular immune response was marked by a higher density of CD8 Tc1-lymphocytes followed by CD4 Th1-lymphocytes producing mainly IFN-γ. The data obtained in the present study suggest that antigen-presenting cells play an important role in the in situ immune response through the production of proinflammatory cytokines, directing the cellular immune response preferentially to the Th1 and Tc1 types in NUCL caused by L. (L.) infantum chagasi.

Leishmania (L.) infantum chagasi isolated from skin lesions of patients affected by non-ulcerated cutaneous leishmaniasis lead to visceral lesion in hamsters.

In Central America, Leishmania (L.) infantum chagasi infection causes visceral leishmaniasis (VL) and non-ulcerated cutaneous leishmaniasis (NUCL). The aim of the present study was to evaluate the course of an experimental infection in hamsters caused by L. (L.) infantum chagasi isolated from patients affected by NUCL compared with a strain isolated from a patient with VL. Stationary phase parasites in culture were inoculated through subcutaneous and intraperitoneal routes in hamsters. Following the post-infection times, a histopathological study, parasite load and cytokine determination in skin from the cutaneous inoculation site and viscera were performed. Animals subcutaneously infected with the different strains did not develop macroscopic lesions at the inoculation site, and the histopathological changes in the dermis were very slight. Regarding the histopathological study of the viscera, we observed the portal mononuclear inflammatory infiltrate, the presence of nodules in the hepatic parenchyma and the proliferation of macrophages in the spleen, which increased over the infection course. Overall, the parasite load in the liver and spleen and in the total IgG titres in the sera of infected hamster showed an increase with the time of infection, regardless of the route of inoculation. Regarding cellular immunity, we did not observe an increase or decrease in pro- and anti-inflammatory cytokines compared to the healthy control, except for IL-10, which was evident in the infected animals. The data showed that strains isolated from NUCL cause visceral lesions in the hamsters regardless of the route of inoculation, and they were similar to parasites isolated from VL humans.

Visceral Leishmaniasis Urbanization in the Brazilian Amazon Is Supported by Significantly Higher Infection Transmission Rates Than in Rural Area.

This was an open cohort prospective study (2016−2018) that analyzed the prevalence and incidence rates of human Leishmania (L.) infantum chagasi-infection and the evolution of their clinical-immunological profiles in distinct urban and rural scenarios of American visceral leishmaniasis (AVL) in Pará State, in the Brazilian Amazon. These infection profiles were based on species-specific DTH/IFAT-IgG assays and clinical evaluation of infected individuals, comprising five profiles: three asymptomatic, Asymptomatic Infection [AI], Subclinical Resistant Infection [SRI], and Indeterminate Initial Infection [III]; and two symptomatic, Subclinical Oligosymptomatic Infection [SOI] and Symptomatic Infection [SI = AVL]. The two distinct scenarios (900 km away) were the urban area of Conceição do Araguaia municipality and the rural area of Bujaru municipality in the southeast and northeast of Pará State. Human populations were chosen based on a simple convenience sampling design (5−10% in each setting), with 1723 individuals (5.3%) of the population (32,464) in the urban area and 1568 individuals (8.9%) of the population (17,596) in the rural one. A serological survey (IFAT-IgG) of canine infection was also performed in both scenarios: 195 dogs in the urban area and 381 in the rural one. Prevalence and incidence rates of human infection were higher in the urban area (20.3% and 13.6/100 person-years [py]) than in the rural setting (14.1% and 6.8/100-py). The AI profile was the most prevalent and incident in both urban (13.4% and 8.1/100-py) and rural (8.3% and 4.2/100-py) scenarios, but with higher rates in the former. An III profile case evolved to SOI profile after four weeks of incubation and another to SI (=AVL) after six. The prevalence of canine infection in an urban setting (39.2%) was also higher (p < 0.05) than that (32%) in the rural zone. AVL urbanization in Pará State, in the Brazilian Amazon, has led to infection rates significantly higher than those in rural sites, requiring more intense control measures.

Leishmania amazonensis from distinct clinical forms/hosts has polymorphisms in Lipophosphoglycans, displays variations in immunomodulatory properties and, susceptibility to antileishmanial drugs.

Lipophosphoglycan (LPG), the major Leishmania glycoconjugate, induces pro-inflammatory/immunosuppressive innate immune responses. Here, we evaluated functional/biochemical LPG properties from six Leishmania amazonensis strains from different hosts/clinical forms. LPGs from three strains (GV02, BA276, and LV79) had higher pro-inflammatory profiles for most of the mediators, including tumor necrosis factor alpha and interleukin 6. For this reason, glycoconjugates from all strains were biochemically characterized and had polymorphisms in their repeat units. They consisted of three types: type I, repeat units devoid of side chains; type II, containing galactosylated side chains; and type III, containing glucosylated side chains. No relationship was observed between LPG type and the pro-inflammatory properties. Finally, to evaluate the susceptibility against antileishmanial agents, two strains with high (GV02, BA276) and one with low (BA336) pro-inflammatory activity were selected for chemotherapeutic tests in THP-1 cells. All analyzed strains were susceptible to amphotericin B (AmB) but displayed various responses against miltefosine (MIL) and glucantime (GLU). The GV02 strain (canine visceral leishmaniasis) had the highest IC50 for MIL (3.34 µM), whereas diffuse leishmaniasis strains (BA276 and BA336) had a higher IC50 for GLU (6.87-12.19 mM). The highest IC50 against MIL shown by the GV02 strain has an impact on clinical management. Miltefosine is the only drug approved for dog treatment in Brazil. Further studies into drug susceptibility of L. amazonensis strains are warranted, especially in areas where dog infection by this species overlaps with those caused by Leishmania infantum.

Histological and neuronal changes in the duodenum of hamsters infected with Leishmania (Leishmania) infantum.

Visceral leishmaniasis is a neglected tropical disease caused by parasites belonging to the Leishmania genus that infect macrophages in different tissues such as the spleen, liver, lymph nodes, bone marrow, and intestine. Therefore, this study aimed to investigate the integrity of the intestinal tract and the nitrergic (NADPH-dp) and metabolically active (NADH-dp) myenteric neurons of the duodenum of golden hamsters infected with L. (L.) infantum. Therefore, thirty golden hamsters were divided into six groups (n = 5); three of them were infected with 2 × 107 promastigote forms of L. (L.) infantum by intraperitoneal route (Infected Group - IG) and three were inoculated with saline solution (control group - CG). After 30, 60 and 90 days post-infection (DPI) infected animals were euthanized and the liver, spleen and duodenum were collected to analyze tissue parasitism. The duodenum was processed using usual histological techniques to analyze the main changes that occurred during infection and histochemical techniques to phenotype myenteric neurons. Amastigote forms were observed in the spleen, liver, and duodenum during all experimental periods, and tissue parasitism in these organs increased significantly over time. At 30 DPI, reduction in muscle tunic, increase in the total intestinal wall and the number of goblet cells PAS+ was observed. At 60 DPI, an increase in intestinal crypts and intraepithelial lymphocytes was observed, and a reduction in intestinal villi was observed at 90 DPI, along with an increase in crypt size. Regarding neurons, an increase in the density of the NADPH-dp population was observed at 30 DPI, but at 60 and 90 DPI a significant reduction of this population was observed. In general, infection progression was observed to cause significant morphofunctional changes in the duodenum of infected hamsters.

Absolute Configuration and Antileishmanial Activity of (-)-Cyclocolorenone Isolated from Duguetia lanceolata (Annonaceae).

BACKGROUND: The fractionation of the n-hexane phase of the EtOH extract from the leaves of Duguetia lanceolata (Annonaceae) led to the identification of the sesquiterpene (-)-cyclocolorenone. OBJECTIVES: Chemical characterization, including determination of the absolute stereochemistry, and in vitro evaluation of antileishmanial activity of the sesquiterpene (-)-cyclocolorenone, isolated from D. lanceolata, were carried out. METHODS: (-)-Cyclocolorenone was isolated from D. lanceolata leaves using different chromatographic steps and its structure was defined by analysis of NMR and ESI-HRMS data. Additionally, the absolute configuration of (-)-cyclocolorenone was ambiguously assigned by means of vibrational circular dichroism (VCD). Antileishmanial activity of (-)-cyclocolorenone was evaluated on promastigote and amastigote forms of Leishmania (Leishmania) amazonensis. The integrity of the cell membrane of L. (L.) amazonensis was analyzed using the SYTOX green probe. RESULTS: (-)-(1R,6S,7R,10R)-Cyclocolorenone displayed activity against promastigotes and amastigotes forms of L. (L.) amazonensis with IC50 of 4.54 and 28.44 µM, respectively. Furthermore, this compound was non-toxic in J774 macrophage cells (CC50 > 458.71 µM) with a selectivity index > 100 (promastigotes) and > 32.2 (amastigotes). Additionally, (-)-cyclocolorenone was observed to target the parasite cell membrane. CONCLUSION: Obtained data suggested that (-)-cyclocolorenone, in which absolute configuration was determined, can be considered as a scaffold for the development of new drugs for the treatment of leishmaniasis.

Mechanistic Insights into the Leishmanicidal and Bactericidal Activities of Batroxicidin, a Cathelicidin-Related Peptide from a South American Viper (Bothrops atrox).

Snake venoms are important sources of bioactive molecules, including those with antiparasitic activity. Cathelicidins form a class of such molecules, which are produced by a variety of organisms. Batroxicidin (BatxC) is a cathelicidin found in the venom of the common lancehead (Bothrops atrox). In the present work, BatxC and two synthetic analogues, BatxC(C-2.15Phe) and BatxC(C-2.14Phe)des-Phe1, were assessed for their microbicidal activity. All three peptides showed a broad-spectrum activity on Gram-positive and -negative bacteria, as well as promastigote and amastigote forms of Leishmania (Leishmania) amazonensis. Circular dichroism (CD) and nuclear magnetic resonance (NMR) data indicated that the three peptides changed their structure upon interaction with membranes. Biomimetic membrane model studies demonstrated that the peptides exert a permeabilization effect in prokaryotic membranes, leading to cell morphology distortion, which was confirmed by atomic force microscopy (AFM). The molecules considered in this work exhibited bactericidal and leishmanicidal activity at low concentrations, with the AFM data suggesting membrane pore formation as their mechanism of action. These peptides stand as valuable prototype drugs to be further investigated and eventually used to treat bacterial and protozoal infections.

Macrophage Polarization in the Skin Lesion Caused by Neotropical Species of Leishmania sp.

Macrophages play important roles in the innate and acquired immune responses against Leishmania parasites. Depending on the subset and activation status, macrophages may eliminate intracellular parasites; however, these host cells also can offer a safe environment for Leishmania replication. In this sense, the fate of the parasite may be influenced by the phenotype of the infected macrophage, linked to the subtype of classically activated (M1) or alternatively activated (M2) macrophages. In the present study, M1 and M2 macrophage subsets were analyzed by double-staining immunohistochemistry in skin biopsies from patients with American cutaneous leishmaniasis (ACL) caused by L. (L.) amazonensis, L. (V.) braziliensis, L. (V.) panamensis ,and L. (L.) infantum chagasi. High number of M1 macrophages was detected in nonulcerated cutaneous leishmaniasis (NUCL) caused by L. (L.) infantum chagasi (M1 = 112 ± 12, M2 = 43 ± 12 cells/mm2). On the other side, high density of M2 macrophages was observed in the skin lesions of patients with anergic diffuse cutaneous leishmaniasis (ADCL) (M1 = 195 ± 25, M2 = 616 ± 114), followed by cases of localized cutaneous leishmaniasis (LCL) caused by L. (L.) amazonensis (M1 = 97 ± 24, M2 = 219 ± 29), L. (V.) panamensis (M1 = 71 ± 14, M2 = 164 ± 14), and L. (V.) braziliensis (M1 = 50 ± 13, M2 = 53 ± 10); however, low density of M2 macrophages was observed in NUCL. The data presented herein show the polarization of macrophages in skin lesions caused by different Leishmania species that may be related with the outcome of the disease.

Molecular tools confirm natural Leishmania (Viannia) guyanensis/L. (V.) shawi hybrids causing cutaneous leishmaniasis in the Amazon region of Brazil.

Seven isolates from patients with American cutaneous leishmaniasis in the Amazon region of Brazil were phenotypically suggestive of Leishmania (Viannia) guyanensis/L. (V.) shawi hybrids. In this work, two molecular targets were employed to check the hybrid identity of the putative hybrids. Heat shock protein 70 (hsp70) gene sequences were analyzed by three different polymerase chain reaction (PCR) approaches, and two different patterns of inherited hsp70 alleles were found. Three isolates presented heterozygous L. (V.) guyanensis/L. (V.) shawi patterns, and four presented homozygous hsp70 patterns involving only L. (V.) shawi alleles. The amplicon sequences confirmed the RFLP patterns. The high-resolution melting method detected variant heterozygous and homozygous profiles. Single-nucleotide polymorphism genotyping/cleaved amplified polymorphic site analysis suggested a higher contribution from L. (V.) guyanensis in hsp70 heterozygous hybrids. Additionally, PCR-RFLP analysis targeting the enzyme mannose phosphate isomerase (mpi) gene indicated heterozygous and homozygous cleavage patterns for L. (V.) shawi and L. (V.) guyanensis, corroborating the hsp70 findings. In this communication, we present molecular findings based on partial informative regions of the coding sequences of hsp70 and mpi as markers confirming that some of the parasite strains from the Brazilian Amazon region are indeed hybrids between L. (V.) guyanensis and L. (V.) shawi.

Nanoemulsified Butenafine for Enhanced Performance against Experimental Cutaneous Leishmaniasis.

The production of ergosterol lipid involves the activity of different enzymes and is a crucial event for the Leishmania membrane homeostasis. Such enzymes can be blocked by azoles and allylamines drugs, such as the antifungal butenafine chloride. This drug was active on parasites that cause cutaneous and visceral leishmaniasis. Based on the leishmanicidal activity of butenafine chloride and considering the absence of reports about the therapeutic potential of this drug in cutaneous leishmaniasis, the present work is aimed at analyzing the efficacy of butenafine formulated in two different topical delivery systems, the self-nanoemulsifying drug delivery systems (BUT-SNEDDS) and in a SNEDDS-based nanogel (BUT-SNEDDS gel) as well as in the free form in experimental cutaneous leishmaniasis. Physical studies showed that both formulations were below 300 nm with low polydispersity (<0.5) and good colloidal stability (around -25 mV). Increased steady-state flux was reported for nanoenabled butenafine formulations with reduced lag time in Franz cell diffusion assays across Strat-M membranes. No toxic or inflammatory reactions were detected in animals treated with BUT-SNEDDS, BUT-SNEDDS gel, or butenafine. Animals topically treated with butenafine (free or nanoformulated) showed small dermal lesions and low tissue parasitism. Furthermore, BUT-SNEDD gel and butenafine presented similar efficacy than the standard drug Glucantime given by the intralesional route. Increased levels of IFN-γ were observed in animals treated with BUT-SNEDDS gel or butenafine. Based on these data, the antifungal drug butenafine chloride can be considered an interesting repurposed drug for the treatment of cutaneous leishmaniasis.

Chromosomal segments may explain the antibody response cooperation for canine leishmaniasis pathogenesis.

Visceral leishmaniasis (VL) is marked by hyperactivation of a humoral response secreting high quantity of immunoglobulins (Igs) that are inaccessible to intracellular parasites. Here we investigated the contributions of the antibody response to the canine leishmaniasis pathogenesis. Using correlation and genome-wide association analysis, we investigated the relationship of anti-Leishmania infantum immunoglobulin classes levels with parasite burden, clinical response, renal/hepatic biochemical, and oxidative stress markers in dogs from endemic areas of VL. Immunoglobulin G (IgG) and IgA were positively correlated with parasite burden on lymph node and blood. Increased IgG, IgA and IgE levels were associated with severe canine leishmaniasis (CanL) whereas IgM was elevated in uninfected exposed dogs. Correlations of IgM, IgG and IgA with creatinine, urea, AST and ALT levels in the serum were suggested an involvement of those Igs with renal and hepatic changes. The correlogram of oxidative radicals and antioxidants revealed a likely relationship of IgM, IgG and IgA with oxidative stress and lipid peroxidation in the blood, suggested as mechanisms mediating tissue damage and CanL worsening. The gene mapping on chromosomal segments associated with the quantitative variation of immunoglobulin classes identified genetic signatures involved with reactive oxygen species generation, phagolysosome maturation and rupture, free iron availability, Th1/Th2 differenciation and, immunoglobulin clearance. The findings demonstrated the roles of the antibody response as resistance or susceptibility markers and mediators of CanL pathogenesis. In addition we pinpointed candidate genes as potential targets for the therapy against the damage caused by exacerbated antibody response and parasitism in VL.

Lipophosphoglycans from dermotropic Leishmania infantum are more pro-inflammatory than those from viscerotropic strains.

Although Leishmania infantum is well-known as the aethiological agent of visceral leishmaniasis (VL), in some Central American countries it may cause atypical non-ulcerated cutaneous leishmaniasis (NUCL). However, the mechanisms favoring its establishment in the skin are still unknown. Lipophosphoglycan (LPG) is the major Leishmania multivirulence factor involved in parasite-host interaction. In the case of viscerotropic L. infantum, it causes an immunosuppression during the interaction with macrophages. Here, we investigated the biochemical and functional roles of LPGs from four dermotropic L. infantum strains from Honduras during in vitro interaction with murine macrophages. LPGs were extracted, purified and their repeat units analysed. They did not have side chains consisting of Gal(ß1,4)Man(α1)-PO4 common to all LPGs. Peritoneal macrophages from BALB/c and C57BL/6 were exposed to LPG for nitric oxide (NO) and cytokine (TNF-α and, IL-6) production. LPGs from dermotropic strains from Honduras triggered higher NO and cytokine levels compared to those from viscerotropic strains. In conclusion, LPGs from dermotropic strains are devoid of side-chains and exhibit high pro-inflammatory activity.

LEISHMANICIDAL ACTIVITY in vivo OF A MILTEFOSINE DERIVATIVE IN Mesocricetus auratus.

Visceral leishmaniasis (VL) is a chronic and systemic disease; if untreated, it can cause death in a large number of cases. The therapy is based on the use of antimonials, which have been used for over 50 years. However, cases of resistance have been reported in some countries. In this context, miltefosine (MIL) was introduced to treat antimonial unresponsive cases. Nonetheless, in recent years MIL unresponsive and relapse cases of VL have increasingly been reported. In the current study, the therapeutic potential of compound 5-(4-(3-methanesulfonatepropyl)-1H-1,2,3-triazol-1-yl)dodecyl methanesulfonate (C11), an MIL derivative, was assessed in an experimental VL hamster model. For this purpose, golden hamsters (Mesocricetus auratus) were infected with Leishmania (L.) infantum chagasi and treated daily for 10 days with C11 and MIL administered orally; in addition, Glucantime (GLU), peritoneal route, were administered at 15, 10, 50 mg/kg body weight/day, respectively. Twenty four hours after the end of treatment the animals were euthanatized; and the specimens were collected to evaluate the relative mRNA expression of cytokines IFN-γ, TNF-α, IL-17, TGF-ß, IL-4 and IL-10 in fragments of the spleen and liver; moreover, the parasitism in these organs was evaluated as well as the main histopathological alterations. The C11-treated animals showed greater expression of IL-17 and TNF-α cytokines and reduced expression of IL-10 in the spleen in comparison to the infected untreated group (UTG) (p <0.05). The C11 and GLU groups showed a significant reduction in the IgG levels in comparison to the UTG group (p <0.05). Moreover, the C11-treated animals had fewer parasites in the spleen than the UTG animals (reduction of 95.9%), as well as a greater preservation of white pulp architecture in the spleen than the UTG, GLU and MIL groups (p <0.05). For the liver, the animals from the C11 and MIL groups showed a significant increase in TNF-α relative expression in comparison to the UTG animals, which would explain the increase in the number of granulomas and the reduction in the parasitic load (p <0.05). Combined, these findings indicate that C11 is an interesting compound that should be considered for the development of new drugs against VL, mainly due to its leishmanicidal effect and immunostimulating action.

Antileishmanial activity and ultrastructural changes of related tetrahydrofuran dineolignans isolated from Saururus cernuus L. (Saururaceae).

OBJECTIVE: This work describes the isolation of anti-Leishmania amazonensis metabolites from Saururus cernuus (Saururaceae). Additionally, ultrastructural changes in promastigotes were evidenced by electron microscopy. METHODS: The MeOH extract from the leaves of S. cernuus was subjected to bioactivity-guided fractionation. Anti-L. amazonensis activity of purified compounds was performed in vitro against promastigote and amastigote forms. KEY FINDINGS: Bioactivity-guided fractionation of the MeOH extract from the leaves of S. cernuus afforded two related tetrahydrofuran dineolignans: threo,threo-manassantin A (1) and threo,erythro-manassantin A (2). Compounds 1 and 2 displayed activity against promastigotes (EC50 of 35.4 ± 7.7 and 17.6 ± 4.2 µm, respectively) and amastigotes (EC50 of 20.4 ± 1.9 and 16.0 ± 1.1 µm, respectively), superior to that determined for the positive control miltefosine (EC50 of 28.7 ± 3.5 µm). Reduced cytotoxicity for host cells was observed for both compounds. Additionally, ultrastructural changes in promastigotes leading to an alteration of structural morphology were observed, as evidenced by electron microscopy. Furthermore, these compounds altered the morphology and physiology of the plasmatic membrane of L. amazonensis. CONCLUSIONS: The obtained results indicated that dineolignans 1 and 2 could be considered as a scaffold for the design of novel and selective drug candidates for the treatment of leishmaniasis.

Antileishmanial activity and ultrastructural changes of sesquiterpene lactones isolated from Calea pinnatifida (Asteraceae).

Bioactivity-guided fractionation of antileishmanial active CH2Cl2 phase of MeOH extract from leaves of Calea pinnatifida led to isolation of two sesquiterpene lactones calein C (1) and calealactone C (2), which structures were stablished on the basis of spectroscopic analysis. Compounds 1 and 2 displayed potent activity against Leishmania amazonensis promastigotes with EC50 of 1.7 and 4.6 µg mL-1, respectively. Compound 2 presented low cytotoxicity for J774 macrophages and displayed activity against amastigote forms of L. amazonensis similar to miltefosine with CC50 values of 31.73 and 27.18 µg mL-1, respectively. Additionally, compounds 1 and 2 caused ultrastructural changes in promastigotes leading to a loss of their classical structural morphology, as evidenced by electron microscopy. Also compound 2 decreased the mitochondria membrane potential. To the best of our knowledge, this is the first occurrence of 1 and 2 in C. pinnatifida. The results obtained highlighted the importance of studying sesquiterpene lactones isolated from Calea pinnatifida in terms of antileishmanial activity, in order to understand the mechanism of action of the isolated compounds in promastigotes forms of L. amazonensis.

Effect of phospholipase A2 inhibitors during infection caused by Leishmania (Leishmania) amazonensis.

BACKGROUND: Lipid metabolites play an important role in parasite differentiation and virulence. Studies have revealed that Leishmania sp. uses prostaglandins to evade innate barriers, thus enabling the parasites to survive inside immune cells. Despite the role of the enzyme Phospholipase A2 (PLA2) in prostaglandins production, few studies have investigated the role of parasite PLA2 during the interaction between L. (L.) amazonensis and the host (in vitro and in vivo) immune cells. METHODS: In the present work, the leishmanicidal effect of PLA2 inhibitors, methyl arachidonyl fluorophosphonate (MAFP), bromoenol lactone (BEL) and aristolochic acid (AA) were investigated in vitro (promastigote and intracellular amastigote forms of L. (L.) amazonensis) and during in vivo infection using BALB/c mice. RESULTS: The aforementioned inhibitors were deleterious to promastigote and amastigote forms of the L. (L.) amazonensis and were non-toxic to peritoneal macrophages from BALB/c mice. L. (L.) amazonensis-infected BALB/c mice treated with the inhibitor BEL presented decreased lesion size and skin parasitism; however, BEL treatment induced hepatotoxicity in BALB/c mice. CONCLUSIONS: Results presented herein suggested that PLA2 inhibitors altered L. (L.) amazonensis viability. In spite of liver toxicity, treatment with BEL was the most selective compound in vitro, as well in vivo, resulting in lower skin parasitism in the infected mice. These findings corroborate the role of PLA2 in parasite virulence and maintenance in vertebrate hosts, and suggest that molecules structurally related to BEL should be considered when planning compounds against Leishmania sp.

Synthesis, leishmanicidal activity, structural descriptors and structure-activity relationship of quinoline derivatives.

AIM: Considering the epidemiology of leishmaniasis, the emergence of resistant parasites to the approved drugs, and severe clinical manifestations, the development of novel leishmanicidal molecules has become of considerable importance. RESULTS: In this work, three commercially available and 19 synthesized quinoline derivatives were evaluated against promastigote and amastigote forms of Leishmania (Leishmania) amazonensis. In addition, structural parameters and molecular electrostatic potentials were obtained by theoretical calculations, allowing statistical (principal component analyses and hierarchical cluster analyses) and comparative (molecular electrostatic potentials vs leishmanicidal activities) studies, respectively. CONCLUSION: Principal component analyses and hierarchical cluster analyses suggested volume and polar surface area as possible structural descriptors for the leishmanicidal activity. Furthermore, a comparison between molecular electrostatic potentials and leishmanicidal activities afforded a reasonable structure-activity relationship.