Dioclea violacea lectin has potent in vitro leishmanicidal activity against Leishmania infantum via carbohydrate recognition domain.

Despite promising advancements in leishmaniasis treatment, existing therapies often face limitations and significant side effects, stimulating the search for novel therapeutic alternatives. In this context, lectins, such as DVL extracted from Dioclea violacea seeds, have emerged as potential candidates due to their diverse biological activities. This study represents the first investigation of the leishmanicidal potential of DVL in vitro against Leishmania infantum. Our results demonstrate that DVL exhibits a leishmanicidal effect (IC50/24 h = 49.37 µg/mL or 2 µM), binding to glycans on L. infantum. Fluorescence assays revealed that DVL can induce the production of reactive oxygen species (ROS) and cause damage to the parasite's membrane. DVL demonstrated a modulating effect when combined with amphotericin B and glucantime, enhancing the activity of these drugs by 40 % and 80 %, respectively. It also showed no cytotoxicity in Raw 264.7 cells and was able to override the toxic effect of amphotericin B on cells and reduce the survival rate of macrophages infected with amastigote forms, as well as their percentage of infection by 31 %. Therefore, DVL shows promise as a treatment for visceral leishmaniasis caused by L. infantum. Our findings provide valuable insights for future therapeutic development targeting leishmania glycans.

Microemulsions strongly promoted the activity of α-bisabolol against different Leishmania species and its skin permeation.

This study aimed to develop microemulsions (MEs) containing α-bisabolol for the topical treatment of cutaneous leishmaniasis (CL). Initially, pseudoternary phase diagrams were developed using α-bisabolol as the oil phase, Eumulgin® CO 40 as the surfactant, Polymol® HE as the co-surfactant, and distilled water as the aqueous phase. Two transparent liquid systems (TLS) containing 5% of α-bisabolol were selected and characterized (F5E25 and F5EP25). Next, skin permeation and retention assays were performed using Franz cells. The interaction of the formulation with the stratum corneum (SC) was evaluated using the FTIR technique. The cytotoxicity was evaluated in murine peritoneal macrophages. Finally, the antileishmanial activity of microemulsions was determined in promastigotes and amastigotes of L. amazonensis (strain MHOM/BR/77/LTB 0016). As a result, the selected formulations showed isotropy, nanometric size (below 25 nm), Newtonian behavior and pH ranging from 6.5 to 6.9. The MEs achieved a 2.5-fold increase in the flux and skin-permeated amount of α-bisabolol. ATR-FTIR results showed that microemulsions promoted fluidization and extraction of lipids and proteins of the stratum corneum, increasing the diffusion coefficient and partition coefficient of the drug in the skin. Additionally, F5E25 and F5EP25 showed higher activity against promastigotes (IC50 13.27 and 18.29, respectively) compared to unencapsulated α-bisabolol (IC50 53.8). Furthermore, F5E25 and F5EP25 also showed antileishmanial activity against intracellular amastigotes of L. amazonensis, with IC50 50 times lower than free α-bisabolol and high selectivity index (up to 15). Therefore, the systems obtained are favorable to topical administration, with significant antileishmanial activity against L. amazonensis promastigotes and amastigotes, being a promising system for future in vivo trials.

Targeting Leishmania Promastigotes and Amastigotes Forms through Amino Acids and Peptides: A Promising Therapeutic Strategy.

Millions of people worldwide are affected by leishmaniasis, caused by the Leishmania parasite. Effective treatment is challenging due to the biological complexity of the parasite, drug toxicity, and increasing resistance to conventional drugs. To combat this disease, the development of specific strategies to target and selectively eliminate the parasite is crucial. This Review highlights the importance of amino acids in the developmental stages of Leishmania as a factor determining whether the infection progresses or is suppressed. It also explores the use of peptides as alternatives in parasite control and the development of novel targeted treatments. While these strategies show promise for more effective and targeted treatment, further studies to address the remaining challenges are imperative.

Highly selective alkylcoumarates and diterpenoids isolated from leaves of Baccharis quitensis: a search for new hit compounds against Trypanosoma cruzi.

In the present work, the hexane extract of aerial parts of Baccharis quitensis Kunth. was subjected to chromatographic fractionation to afford two alkyl phenylpropanoids: n-docosyl (E)-p-coumarate (1) and n-tetracosyl (E)-p-coumarate (2) as well as five diterpenes: ent-kaurenoic acid (3), grandifloric acid (4), 15ß-senecioyl-oxy-ent-kaur-16-en-19-oic acid (5), and 15-oxo-ent-kaurenoic acid (6). Using an ex-vivo assay with macrophages infected with Trypanosoma cruzi, compounds 1 and 2 demonstrated high potency against intracellular amastigotes, with EC50 values of 7.5 and 6.9 µM, respectively. Compound 6 revealed a moderate potency against T. cruzi, with an EC50 of 25.6 µM, and compounds 3-5 showed no effectiveness. Additionally, compounds 1-6 compounds presented no toxicity for mammalian cells to the highest tested concentration of 200 µM. Based on potency and the selectivity indexes of 1, 2 and 6, these compounds could be future candidates for optimisation studies for the design of prototypes against Chagas disease.

Highly selective alkylcoumarates and diterpenoids isolated from leaves of Baccharis quitensis: a search for new hit compounds against Trypanosoma cruzi

In the present work, the hexane extract of aerial parts of Baccharis quitensis Kunth. was subjected to chromatographic fractionation to afford two alkyl phenylpropanoids: n-docosyl (E)-p-coumarate (1) and n-tetracosyl (E)-p-coumarate (2) as well as five diterpenes: ent-kaurenoic acid (3), grandifloric acid (4), 15β-senecioyl-oxy-ent-kaur-16-en-19-oic acid (5), and 15-oxo-ent-kaurenoic acid (6). Using an ex-vivo assay with macrophages infected with Trypanosoma cruzi, compounds 1 and 2 demonstrated high potency against intracellular amastigotes, with EC50 values of 7.5 and 6.9 µM, respectively. Compound 6 revealed a moderate potency against T. cruzi, with an EC50 of 25.6 µM, and compounds 3–5 showed no effectiveness. Additionally, compounds 1–6 compounds presented no toxicity for mammalian cells to the highest tested concentration of 200 µM. Based on potency and the selectivity indexes of 1, 2 and 6, these compounds could be future candidates for optimisation studies for the design of prototypes against Chagas disease.

First reported case of leishmaniasis in a cat in Trinidad and Tobago.

A 3-year-old, female, domestic shorthair cat, was presented to the Veterinary Teaching Hospital at the School of Veterinary Medicine (SVM), Trinidad and Tobago for a swollen nose, and multiple, variably sized small masses on both ears. The initial diagnostic tests included a CBC, serum biochemistry profile, cytological evaluation of masses on the ear and nose, and FeLV/FIV testing. The CBC and biochemistry results were unremarkable except for a hyperproteinaemia and hyperglobulinemia. Cytology of the nose and ear lesions revealed mixed inflammation and high numbers of intracellular and extracellular organisms consistent with Leishmania amastigotes. The cat was FeLV/FIV negative. Histopathology and Leishmania IFA and PCR analysis were subsequently performed, confirming the Leishmania diagnosis. The PCR, DNA sequencing and phylogenetic tree analyses identified L. amazonensis. This is the first reported case of L. amazonensis infection in a domestic animal in Trinidad with molecular characterization indicating it exists in the region and is likely being transmitted by sandflies.

Poliartrite em cão por Leishmaniose - tratamento com miltefosina / Polyarthritis in a dog due to leishmaniasis - treatment with miltefosine

Background: Canine visceral leishmaniasis causes several clinical signs, such as lymphadenomegaly, exfoliative dermatitis, ulcerative skin lesions, and lameness. The most commonly reported locomotor changes are claudication, edema, arthralgia, joint stiffness, and muscle atrophy. Radiographic exam revealed cortical and medullary destruction, increase, or decrease in medullary opacity, proliferative periosteal reaction, osteolysis, collapse of joint spaces and soft tissue edema are observed. The aim of this report is to describe the clinical and radiographic evolution of a case of erosive polyarthritis associated with leishmaniasis in a dog before, during and after treatment with miltefosine. Case: A 7-month-old mixed-breed dog was attended due pain and limited mobility. In the orthopedic evaluation, joint swelling, stiffness, and increased pain sensitivity of the four limbs, as well as neck stiffness, were noted. Radiographic examination showed joint changes compatible with edema, with increased volume and radiopacity of the soft tissues adjacent to the joints. The segments of the patient's spine showed more severe bone alterations, the cervical spine being one of the most affected regions, with multiple bone proliferations throughout the vertebral body, especially in the ventral portion (spondylosis), compatible with polyarthritis due to leishmaniasis. Due to the suspicion, lymph node and spleen cytology was performed, confirming the diagnosis. Hematological examination revealed anemia, leukopenia due to lymphopenia and thrombocytopenia in addition to increased AST (79,4 U/L; reference: 6,2 - 13 U/L), creatine kinase (517,6 U/L; reference: 1,5 - 28,4 U/L), lactate dehydrogenase (688,4 IU/L; reference: 45 - 233 IU/L) and hyperproteinemia (7,34 g/dL; reference: 5,4 - 7,1 g/dL). Treatment with miltefosine, allopurinol, domperidone, prednisone, gabapentin and dipyrone was started. Reassessments were performed monthly for 3 consecutive months. Hematological examinations showed improvement, with resolution of anemia and thrombocytopenia, and a marked decrease in creatine kinase values. Thus, it is evident that the dog did not develop liver or kidney changes during treatment. During the treatment and monitoring in this period, the dog had a clinical improvement, which started to walk without pain. In addition, joint swellings were no longer present, however, there was no improvement in the radiographic evaluation of the joints. Discussion: Clinical signs of the locomotor system are compatible with those described in animals that had osteoarticular manifestations associated with leishmaniasis, such as arthralgia, edema, and joint stiffness. In the present report, treatment with miltefosine associated with allopurinol resulted in an improvement in the clinical picture, and this therapy is therefore promising in dogs with polyarthritis due to leishmaniasis. A case published in human medicine demonstrated the intra-articular absorption capacity of this drug. There is only one study to date that describes the radiographic evolution of a dog with arthritis due to leishmaniasis after treatment with miltefosine and allopurinol. In this case described, the dog reported remained with the osteoarticular lesions after treatment, although clinical improvement was observed, as in our report. The use of miltefosine and allopurinol are in accordance with stage II staging for leishmaniasis. In this study, although there was no improvement in the radiographic examinations, the treatment was effective in the remission of the animal's clinical condition.

Induction of Autophagy by Ursolic Acid Promotes the Elimination of Trypanosoma cruzi Amastigotes From Macrophages and Cardiac Cells.

Chagas disease, caused by the parasite Trypanosoma cruzi, is an infectious illness endemic to Latin America and still lacks an effective treatment for the chronic stage. In a previous study in our laboratory, we established the protective role of host autophagy in vivo during T. cruzi infection in mice and proposed this process as one of the mechanisms involved in the innate immune response against this parasite. In the search for an autophagy inducer that increases the anti-T. cruzi response in the host, we found ursolic acid (UA), a natural pentacyclic triterpene with many biological actions including autophagy induction. The aim of this work was to study the effect of UA on T. cruzi infection in vitro in the late infection stage, when the nests of intracellular parasites are forming, in both macrophages and cardiac cells. To test this effect, the cells were infected with T. cruzi for 24 h and then treated with UA (5-10 µM). The data showed that UA significantly decreased the number of amastigotes found in infected cells in comparison with non-treated cells. UA also induced the autophagy response in both macrophages and cardiac cells under the studied conditions, and the inhibition of this pathway during UA treatment restored the level of infection. Interestingly, LC3 protein, the main marker of autophagy, was recruited around amastigotes and the acidic probe LysoTracker localized with them, two key features of xenophagy. A direct cytotoxic effect of UA was also found on trypomastigotes of T. cruzi, whereas epimastigotes and amastigotes displayed more resistance to this drug at the studied concentrations. Taken together, these data showed that this natural compound reduces T. cruzi infection in the later stages by promoting parasite damage through the induction of autophagy. This action, in addition to the effect of this compound on trypomastigotes, points to UA as an interesting lead for Chagas disease treatment in the future.

Ablation of the P21 Gene of Trypanosoma cruzi Provides Evidence of P21 as a Mediator in the Control of Epimastigote and Intracellular Amastigote Replication.

P21 is an immunomodulatory protein expressed throughout the life cycle of Trypanosoma cruzi, the etiologic agent of Chagas disease. In vitro and in vivo studies have shown that P21 plays an important role in the invasion of mammalian host cells and establishment of infection in a murine model. P21 functions as a signal transducer, triggering intracellular cascades in host cells and resulting in the remodeling of the actin cytoskeleton and parasite internalization. Furthermore, in vivo studies have shown that P21 inhibits angiogenesis, induces inflammation and fibrosis, and regulates intracellular amastigote replication. In this study, we used the CRISPR/Cas9 system for P21 gene knockout and investigated whether the ablation of P21 results in changes in the phenotypes associated with this protein. Ablation of P21 gene resulted in a lower growth rate of epimastigotes and delayed cell cycle progression, accompanied by accumulation of parasites in G1 phase. However, P21 knockout epimastigotes were viable and able to differentiate into metacyclic trypomastigotes, which are infective to mammalian cells. In comparison with wild-type parasites, P21 knockout cells showed a reduced cell invasion rate, demonstrating the role of this protein in host cell invasion. However, there was a higher number of intracellular amastigotes per cell, suggesting that P21 is a negative regulator of amastigote proliferation in mammalian cells. Here, for the first time, we demonstrated the direct correlation between P21 and the replication of intracellular amastigotes, which underlies the chronicity of T. cruzi infection.

ß-carbolines RCC and C5 induce the death of Leishmania amazonensis intracellular amastigotes.

Background: Cutaneous leishmaniasis is caused by Leishmania spp., and its treatment is limited. The ß-carbolines have shown activity against kinetoplastids. Aim: To evaluate the activity and effects of the ß-carbolines, N-{2-[(4,6-bis(isopropylamino)-1,3,5-triazin-2-yl)amino]ethyl}-1-(4-methoxyphenyl)-ß-carboline-3-carboxamide (RCC) and N-benzyl-1-(4-methoxy)phenyl-9H-beta-carboline-3-carboxamide (C5), against L. amazonensis intracellular amastigotes and to suggest their mechanism of action. Methods: We analyzed the activity and cytotoxicity of ß-carbolines and the morphological alterations by electron microscopy. Mitochondrial membrane potential, production nitric oxide, reactive oxygen species, lipidic bodies, autophagic vacuoles and ATP were also evaluated. Results & conclusion: The results showed that RCC and C5 are active against intracellular amastigotes and were able to induce oxidative stress and ultrastructural alterations such as accumulation of lipid bodies and autophagic vacuoles, leading to parasite death.

Histological and immunohistochemical evaluations of the bone marrow from femur and sternal manubrium of dogs reactive for leishmaniasis by DPP® and ELISA tests / Avaliações histológica e imunoistoquímica da medula óssea do fêmur e do manúbrio esternal de cães reagentes para leishmaniose aos testes DPP® e ELISA

As the bone marrow is one of the most organs affected by canine visceral leishmaniasis (CVL), samples from this are frequently taken for parasitological tests, with occurrence of myelodysplastic changes, with consequent anemia, leukopenia, and thrombocytopenia. Thus, this study aimed to investigate the histological and immunohistochemical changes in the bone marrow of the femur and sternal manubrium of dogs reactive for leishmaniasis by DPP® and ELISA tests. For this, thirteen canines from the epidemiological routine for CVL carried out by the Directorate of Zoonosis Surveillance of Goiânia (DVZ), GO, Brazil, were subjected to anatomopathological examination. 46.2% of bone marrow samples from the femur showed a higher proportion of the red series, and 53.9% of bone marrow of the sternal manubrium evidenced a higher proportion of the red series. Also, there were varied macrophage hyperplasia, hemosiderosis, and megakaryocytic emperipolesis. Amastigote forms of Leishmania spp. in the bone marrow of the femur and sternal manubrium to histopathological and immunohistochemical evaluations were observed, with good agreement them, but without difference in the parasite intensity between the bone marrow of these anatomical sites. It was concluded that bone marrow of the femur and sternal manubrium of dogs reactive for leishmaniasis by DPP® and ELISA tests has histological changes resulting from the disease, regardless of the parasite presence or intensity, with macrophage hyperplasia, hemosiderosis, and emperipolesis being the main medullary changes in these animals. Also, the bone marrow of the femur and sternal manubrium are useful anatomical sites for the diagnosis of CVL by direct methods.

Como a medula óssea é um dos órgãos mais acometidos pela leishmaniose visceral canina (LVC), amostras desta são frequentemente colhidas para exames parasitológicos, sendo possível a ocorrência de alterações mielodisplásicas, com consequente anemia, leucopenia e trombocitopenia. Assim, este estudo teve como objetivo investigar alterações histológicas e imunoistoquímicas na medula óssea do fêmur e manúbrio esternal de cães reativos para leishmaniose aos testes DPP® e ELISA. Para isso, 13 caninos da rotina epidemiológica para LVC realizada pela Diretoria de Vigilância de Zoonoses de Goiânia (DVZ), GO, Brasil, foram submetidos ao exame anatomopatológico. 46,2% e 53,9% das amostras de medula óssea do fêmur e do manúbrio esternal apresentaram maior proporção da série vermelha, respectivamente. Além disso, havia variados graus de hiperplasia macrofágica, hemossiderose e emperipolese megacariocítica. Formas amastigotas de Leishmania spp. na medula óssea do fêmur e do manúbrio esternal às avaliações histopatológicas e imunoistoquímicas foram observadas, com boa concordância entre essas, mas sem diferença na intensidade parasitária entre a medula óssea desses sítios anatômicos. Conclui-se que a medula óssea do fêmur e do manúbrio esternal de cães reativos para leishmaniose aos testes DPP® e ELISA apresenta alterações histológicas decorrentes da doença, independente da presença ou intensidade do parasito, sendo hiperplasia de macrófagos, hemossiderose e emperipolese as principais alterações medulares nesses animais. Além disso, a medula óssea do fêmur e do manúbrio esternal compreendem sítios anatômicos úteis ao diagnóstico de LVC por métodos diretos.

Assessment of in vitro and in vivo effect of Quercetin 3-Glucoside, Oxyresveratrol and Quercetin O-Hexoside against Leishmania tropica

Abstract The aim was to scrutinize the in vivo and in vitro activities against Leishmania tropica with compounds of Oxyresveratrol, Quercetin O-Hexoside, and Quercetin 3-Glucoside. The in vitro outcomes against Leishmania were analyzed for 24-48 hours on L. tropica KWH23 promastigotes with compounds materials having 50 - 200 µg/mL concentration with negative control and standard drug Amphotericin B. The compounds were analyzed in L. tropica infected BALB/c mice against Leishmania tropica. The Quercetin 3-Glucoside shows mean inhibition of extracellular promastigotes after 48 hours at 50, 100, 150, 200 µg/mL were 91.02 ± 0.12, 94.50 ± 0.07, 96.15 ± 0.17 and 97.01 ± 0.08 % respectively. In BALB/c mice, the intracellular amastigotes were 91% cured at 200 µg/mL and mean lesion size decreased to 0.41 ± 0.21 mm (p < 0.01). The result shows that Quercetin 3-Glucoside possesses significant anti-leishmanial activity.

Planejamento e desenvolvimento de novos compostos com atividade frente a Leishmania spp: Síntese, avaliação da atividade e toxicidade de derivados 5-nitro-2-furfurilidênicos e estudos de relações estrutura-atividade / Design of new compounds against Leishmania spp: synthesis, biological evaluation and toxicity of 5-nitro-2-furfurylidene derivates and structure-activity relationship studies

A leishmaniose é uma zoonose de ampla distribuição mundial, causada pelos parasitas tripanossomatídeos do gênero Leishmania. Infelizmente, o arsenal terapêutico disponível é precário, mas vê-se crescente o interesse científico pela busca do potencial de derivados nitroheterocíclicos como alternativas terapêuticas. Nesse contexto, este trabalho analisou o potencial de derivados 5-nitro-2-furfurilidênicos contra diferentes cepas de Leishmania, assim como investigou um possível modo de ação para esta classe de nitrocompostos. Para tal, a quimioteca foi sintetizada de acordo com publicações prévias do grupo. O potencial de inibição de crescimento das culturas de promastigotas de L. (L.) infantum (Linf) e L. (L.) major (Lmaj) foi determinado, utilizando miltefosina (MILT) (Linf - IC50: 8,28±0,33 µM), anfotericina B (AMB) (Linf - IC50: 0,02±0,002 µM) e nifurtimox (NFX) (Lmaj - IC50: 3,5±0,09 µM) como referência. A maioria dos compostos apresentaram maior potencial que as referênias, destacando o composto 40 (Linf - IC50: 0,2±0,019 µM/ Lmaj - IC50: 0,087 ± 0,001 µM) como mais eficaz. Contra as formas amastigotas intracelulares, para Linf os compostos 40, 13 e 15 foram mais eficazes em reduzir a carga parasitária dos macrófagos infectados que fármacos de referência. Para Lmajor, o composto 40 (IC50: 0,006 ± 0,0003 µM) foi mais ativo que o NFX (IC50: 2,15 ± 0,01 µM). Também foi determinada a atividade da quimioteca frente a enzima nitrorredutase (NTR1), utilizando cepas de T. brucei superexpressantes de NTR1, e os compostos analisados foram até 18 vezes mais eficazes que à cepa wild-type. Ademais, a partir da análise exploratória de dados por análise de componentes principais (PCA) e de grupamentos hierárquicos (HCA), foi reconhecida a influência das propriedades relacionadas com o equilíbrio hidrófilo-lipófilo e da natureza estérica/geométrica das moléculas para atividade anti-Leishmania

Leishmaniasis is a worldwide zoonosis caused by trypanosomatid parasites of the genus Leishmania. Unfortunately, the available therapeutic arsenal is precarious, but there is growing scientific interest in searching the potential of nitroheterocyclic derivatives as therapeutic alternatives. In this context, this work analyzed the potential of 5-nitro-2-furfurylidene derivatives against different Leishmania strains, as well as investigated the potential mode of action for this nitro compounds class. To this end, the chemolibrary was synthesized according to our group's previous publications. The growth inhibitory potential potential for promastigote cultures of L. (L.) infantum (Linf) and L. (L.) major (Lmaj) was determined using miltefosine (MILT) (Linf - IC50: 8.28±0.33 µM), amphotericin B (AMB) (Linf - IC50: 0.02±0.002 µM) and nifurtimox (NFX) (Lmaj - IC50: 3.5±0.09 µM) as reference. Most of the compounds were more potent than the references, highlighting compound 40 (Linf - IC50: 0.2±0.019 µM/ Lmaj - IC50: 0.087 ± 0.001 µM) as the most effective. Against intracellular amastigote, for Linf, compounds 40, 13 and 15 were more effective in reducing the parasite load of infected macrophages than reference drugs. For Lmajor, compound 40 (IC50: 0.006 ± 0.0003 µM) was more active than NFX (IC50: 2.15 ± 0.01 µM). The activity against nitroreductase (NTR1) enzyme was determined using overexpressing NTR1 mutant T. brucei strains, and the analyzed compounds were up to 18 times more effective than wild-type. Furthermore, exploratory data analysis using principal component analysis (PCA) and hierarchical clustering (HCA) methods were used. The influence of properties related to the hydrophiliclipophilic balance and the steric/geometric nature of the molecules was associated with the anti-Leishmanial activity

Evaluation of the Photodynamic Therapy with Curcumin on L. braziliensis and L. major Amastigotes.

Cutaneous leishmaniasis (CL) is a neglected disease prevalent in tropical countries with the ability to cause skin lesions. Photodynamic therapy (PDT) represents a specific and topical option for the treatment of these lesions. This study evaluated the response of macrophages infected with L. braziliensis and L. major to PDT with curcumin. Curcumin concentrations were evaluated in serial dilutions from 500.0 to 7.8 µg/mL using LED (λ = 450 ± 5 nm), with a light dose of 10 J/cm2. The Trypan blue viability test, ultrastructural analysis by scanning electron microscopy (SEM), mitochondrial polarity by Rhodamine 123 (Rho 123), curcumin internalization by confocal microscopy, and counting of the recovered parasites after the PDT treatment were performed. The lowest concentrations of curcumin (15.6 and 7.8 µg/mL) presented photodynamic inactivation. Cell destruction and internalization of curcumin in both macrophages and intracellular parasites were observed in microscopy techniques. In addition, an increase in mitochondrial membrane polarity and a decrease in the number of parasites recovered was observed in the PDT groups. This study indicates that PDT with curcumin has the potential to inactivate infected macrophages and might act as a basis for future in vivo studies using the parameters herein discussed.

Trypanosoma cruzi extracellular amastigotes engage Rac1 and Cdc42 to invade RAW macrophages.

Cell invasion by Trypanosoma cruzi extracellular amastigotes (EAs) relies significantly upon the host cell actin cytoskeleton. In past decades EAs have been established as a reliable model for phagocytosis inducer in non-phagocytic cells. Our current hypothesis is that EAs engage a phagocytosis-like mechanism in non-professional phagocytic cells; however, the molecular mechanisms in professional phagocytes still remain unexplored. In this work, we evaluated the involvement of Rac1 and Cdc42 in the actin-dependent internalization of EAs in RAW 264.7 macrophages. Kinetic assays showed similar internalization of EAs in unstimulated RAW and non-phagocytic HeLa cells but increased in LPS/IFN-γ stimulated RAW cells. However, depletion of Rac1, Cdc42 or RhoA inhibited EA internalization similarly in both unstimulated and stimulated RAW cells. Overexpression of active, but not the dominant-negative, construct of Rac1 increased EA internalization. Remarkably, for Cdc42, both the active and the inactive mutants decreased EA internalization when compared to wild type groups. Despite that, both Rac1 and Cdc42 activation mutants were similarly recruited to and colocalized with actin at the EA-macrophage contact sites when compared to their native isoforms. Altogether, these results corroborate that EAs engage phagocytic processes to invade both professional and non-professional phagocytic cells providing evidences of converging actin mediated mechanisms induced by intracellular pathogens in both cell types.

The cytostome-cytopharynx complex of intracellular and extracellular amastigotes of Trypanosoma cruzi exhibit structural and functional differences.

Endocytosis in Trypanosoma cruzi is mainly performed through a specialised membrane domain called cytostome-cytopharynx complex. Its ultrastructure and dynamics in endocytosis are well characterized in epimastigotes, being absent in trypomastigotes, that lack endocytic activity. Intracellular amastigotes also possess a cytostome-cytopharynx but participation in endocytosis of these forms is not clear. Extracellular amastigotes can be obtained from the supernatant of infected cells or in vitro amastigogenesis. These amastigotes share biochemical and morphological features with intracellular amastigotes but retain trypomastigote's ability to establish infection. We analysed and compared the ultrastructure of the cytostome-cytopharynx complex of intracellular amastigotes and extracellular amastigotes using high-resolution tridimensional electron microscopy techniques. We compared the endocytic ability of intracellular amastigotes, obtained through host cell lysis, with that of extracellular amastigotes. Intracellular amastigotes showed a cytostome-cytopharynx complex similar to epimastigotes'. However, after isolation, the complex undergoes ultrastructural modifications that progressively took to an impairment of endocytosis. Extracellular amastigotes do not possess a cytostome-cytopharynx complex nor the ability to endocytose. Those observations highlight morpho functional differences between intra and extracellular amastigotes regarding an important structure related to cell metabolism. TAKE AWAYS: T. cruzi intracellular amastigotes endocytose through the cytostome-cytopharynx complex. The cytostome-cytopharynx complex of intracellular amastigotes is ultrastructurally similar to the epimastigote. Intracellular amastigotes, once outside the host cell, disassembles the cytostome-cytopharynx membrane domain. Extracellular amastigotes do not possess a cytostome-cytopharynx either the ability to endocytose.

Leishmania amazonensis response to artemisinin and derivatives.

The worldwide presence of Leishmania parasites increases in the poorest regions. Current leishmaniasis treatments are unsatisfactory due to resistance development, side effects and cost. Herein, we describe the in vitro activity of artemisinin (ART), artemether (ATM), artesunate (ATS) and dihydroartemisinin (DHA) against Leishmania amazonensis. Selected compounds were assayed in the animal model of cutaneous leishmaniasis in BALB/c mice. On intracellular amastigotes, similar activity (p > 0.05) was observed for ART, ATM and ATS (IC50 = 15.0-19.2 µM), which were inferior (p < 0.05) respect to reference endoperoxide ascaridole (IC50 = 11.5 ±â€¯1.0 µM) and superior (p < 0.05) compared with reference drug Glucantime® (IC50 = 30.1 ±â€¯9.0 µM). In contrast, DHA (IC50 = 38.5 ±â€¯4.7 µM) showed higher IC50 values (p < 0.05) than other artemisinins and ascaridole, but similar (p > 0.05) than Glucantime®; while deoxyartemisinin caused smaller inhibition (IC50 = 88.9 ±â€¯5.2 µM). Selectivity indexes of >13, 6, 11 and 1 were obtained for ART, ATM, ATS and DHA, respectively. In addition, the potential effect of ART and ATS was also demonstrated in the murine model, causing a significant reduction (p < 0.05) of the lesion size and parasite load regarding untreated animals and treated with vehicle. Effects of both artemisinins were comparable (p > 0.05) with Glucantime® and ascaridole-treated mice. In particular, artemisinin is recommended to further studies, which could be an advantage over the ascaridole endoperoxide and could be useful in endemic areas of parasite resistance to antimonials.

Use of Natural Products in Leishmaniasis Chemotherapy: An Overview.

Leishmaniasis is an infectious parasitic disease that is caused by protozoa of the genus Leishmania, a member of the Trypanosomatidae family. Leishmaniasis is classified by the World Health Organization as a neglected tropical disease that is responsible for millions of deaths worldwide. Although there are many possible treatments for leishmaniasis, these treatments remain mostly ineffective, expensive, and long treatment, as well as causing side effects and leading to the development of resistance. For novel and effective treatments to combat leishmaniasis, many research groups have sought to utilize natural products. In addition to exhibiting potential as therapeutic compounds, natural products may also contribute to the development of new drugs based on their chemical structures. This review presents the most promising natural products, including crude extracts and isolated compounds, employed against Leishmania spp.

Reactivity of purified and axenic amastigotes as a source of antigens to be used in serodiagnosis of canine visceral leishmaniasis.

Although there is a great diversity of techniques and antigens used in the serodiagnosis of canine visceral leishmaniasis (CVL), total sensitivity and specificity have not yet been found. Since the use of amastigote forms in the indirect immunofluorescence assay has shown an improvement in the specificity of the test for the diagnosis of CVL, the performance of amastigotes forms of L. (L.) infantum chagasi as antigen source were evaluated in automatized ELISA test using crude antigen of axenic amastigote and purified amastigote from spleen of hamster chronically infected comparing with ELISA using total antigen produced with promastigote forms of L. (L.) infantum chagasi. One hundred and fifteen sera from dogs with positive parasitological diagnosis by PCR were used. The animals were classified into 2 groups: symptomatic (n = 67) and asymptomatic (n = 48) animals, in accordance with the clinical signs and laboratory tests were. As control, ninety-four sera from dogs with negative parasitological diagnosis were included. No significant difference was found in sensitivity, specificity, predictive values and accuracy between ELISA using whole antigens produced with both axenic and purified amastigotes in comparison with promastigotes forms. Correlation and concordance between the three total antigens tested in ELISA was observed. According to the similar performance among antigens, data pointed out to use antigen from promastigote forms for diagnosing canine leishmaniasis, especially due the easily in the production, lower cost and the abundance of correlative literature.

Comparing the Antileishmanial Activity of Gold(I) and Gold(III) Compounds in L. amazonensis and L. braziliensis in Vitro.

A series of mononuclear coordination or organometallic AuI /AuIII complexes (1-9) have been comparatively studied in vitro for their antileishmanial activity against promastigotes and amastigotes, the clinically relevant parasite form, of Leishmania amazonensis and Leishmania braziliensis. One of the cationic AuI bis-N-heterocyclic carbenes (3) has low EC50 values (ca. 4 µM) in promastigotes cells and no toxicity in host macrophages. Together with two other AuIII complexes (6 and 7), the compound is also extremely effective in intracellular amastigotes from L. amazonensis. Initial mechanistic studies include an evaluation of the gold complexes' effect on L. amazonensis' plasma membrane integrity.