Antitumor Effect of Açaí (Euterpe oleracea Mart.) Seed Extract in LNCaP Cells and in the Solid Ehrlich Carcinoma Model.

Euterpe oleracea (açaí) fruit has approximately 15% pulp, which is partly edible and commercialized, and 85% seeds. Although açaí seeds are rich in catechins-polyphenolic compounds with antioxidant, anti-inflammatory, and antitumor effects-almost 935,000 tons/year of seeds are discarded as industrial waste. This work evaluated the antitumor properties of E. oleracea in vitro and in vivo in a solid Ehrlich tumor in mice. The seed extract presented 86.26 ± 0.189 mg of catechin/g of extract. The palm and pulp extracts did not exhibit in vitro antitumor activity, while the fruit and seed extracts showed cytotoxic effects on the LNCaP prostate cancer cell line, inducing mitochondrial and nuclear alterations. Oral treatments were performed daily at 100, 200, and 400 mg/kg of E. oleracea seed extract. The tumor development and histology were evaluated, along with immunological and toxicological parameters. Treatment at 400 mg/kg reduced the tumor size, nuclear pleomorphism, and mitosis figures, increasing tumor necrosis. Treated groups showed cellularity of lymphoid organs comparable to the untreated group, suggesting less infiltration in the lymph node and spleen and preservation of the bone marrow. The highest doses reduced IL-6 and induced IFN-γ, suggesting antitumor and immunomodulatory effects. Thus, açaí seeds can be an important source of compounds with antitumor and immunoprotective properties.

A New Strategy for Mapping Epitopes of LACK and PEPCK Proteins of Leishmania amazonensis Specific for Major Histocompatibility Complex Class I.

Leishmaniasis represents a complex of diseases with a broad clinical spectrum and epidemiological diversity, considered a major public health problem. Although there is treatment, there are still no vaccines for cutaneous leishmaniasis. Because Leishmania spp. is an intracellular protozoan with several escape mechanisms, a vaccine must provoke cellular and humoral immune responses. Previously, we identified the Leishmania homolog of receptors for activated C kinase (LACK) and phosphoenolpyruvate carboxykinase (PEPCK) proteins as strong immunogens and candidates for the development of a vaccine strategy. The present work focuses on the in silico prediction and characterization of antigenic epitopes that might interact with mice or human major histocompatibility complex class I. After immunogenicity prediction on the Immune Epitope Database (IEDB) and the Database of MHC Ligands and Peptide Motifs (SYFPEITHI), 26 peptides were selected for interaction assays with infected mouse lymphocytes by flow cytometry and ELISpot. This strategy identified nine antigenic peptides (pL1-H2, pPL3-H2, pL10-HLA, pP13-H2, pP14-H2, pP15-H2, pP16-H2, pP17-H2, pP18-H2, pP26-HLA), which are strong candidates for developing a peptide vaccine against leishmaniasis.

Nitric Oxide Induction in Peritoneal Macrophages by a 1,2,3-Triazole Derivative Improves Its Efficacy upon Leishmania amazonensis In Vitro Infection.

1,2,3-Triazole is one of the most flexible chemical scaffolds broadly used in various fields. Here, we report the antileishmanial activity of 1,2,3-triazole derivatives, the ultrastructural alterations induced by their treatment, and the nitric oxide (NO) modulation effect on their efficacy against Leishmania amazonensis in vitro infection. After the screening of eleven compounds, compound 4 exhibited better results against L. amazonensis promastigotes (IC50 = 15.52 ± 3.782 µM) and intracellular amastigotes (IC50 = 4.10 ± 1.136 µM), 50% cytotoxicity concentration at 84.01 ± 3.064 µM against BALB/c peritoneal macrophages, and 20.49-fold selectivity for the parasite over the cells. Compound 4 induced ultrastructural mitochondrial alterations and lipid inclusions in L. amazonensis promastigotes, upregulated tumor necrosis factor α, interleukin (IL)-1ß, IL-6, IL-12, and IL-10 messenger RNA expressions, and enhanced the NO production, verified by nitrite (p = 0.0095) and inducible nitric oxide synthase expression (p = 0.0049) quantification, which played an important role in its activity against intramacrophagic L. amazonensis. In silico prediction in association with antileishmanial activity results showed compound 4 as a hit compound with promising potential for further studies of new leishmaniasis treatment options.

Antiprotozoal and Antibacterial Activity of Ravenelin, a Xanthone Isolated from the Endophytic Fungus Exserohilum rostratum.

The natural compound ravenelin was isolated from the biomass extracts of Exserohilum rostratum fungus, and its antimicrobial, antiplasmodial, and trypanocidal activities were evaluated. Ravenelin was isolated by column chromatography and HPLC and identified by NMR and MS. The susceptibility of Gram-positive and Gram-negative bacteria strains to ravenelin was determined by microbroth dilution assay. Cytotoxicity was evaluated in hepatocarcinoma cells (HepG2) and BALB/c peritoneal macrophages by using MTT. SYBR Green I-based assay was used in the asexual stages of Plasmodium falciparum. Trypanocidal activity was tested against the epimastigote and intracellular amastigote forms of Trypanosoma cruzi. Ravenelin was active against Gram-positive bacteria strains, with emphasis on Bacillus subtilis (MIC value of 7.5 µM). Ravenelin's antiparasitic activities were assessed against both the epimastigote (IC50 value of 5 ± 1 µM) and the intracellular amastigote forms of T. cruzi (IC50 value of 9 ± 2 µM), as well as against P. falciparum (IC50 value of 3.4 ± 0.4 µM). Ravenelin showed low cytotoxic effects on both HepG2 (CC50 > 50 µM) and peritoneal macrophage (CC50 = 185 ± 1 µM) cells with attractive selectivity for the parasites (SI values > 15). These findings indicate that ravenelin is a natural compound with both antibacterial and antiparasitic activities, and considerable selectivity indexes. Therefore, ravenelin is an attractive candidate for hit-to-lead development.

GC-MS Characterization of Antibacterial, Antioxidant, and Antitrypanosomal Activity of Syzygium aromaticum Essential Oil and Eugenol.

Syzygium aromaticum has a diversity of biological activities due to the chemical compounds found in its plant products such as total phenolic compounds and flavonoids. The present work describes the chemical analysis and antimicrobial, antioxidant, and antitrypanosomal activity of the essential oil of S. aromaticum. Eugenol (53.23%) as the major compound was verified by gas chromatography-mass spectrometry. S. aromaticum essential oil was more effective against S. aureus (MIC 50 µg/mL) than eugenol (MIC 250 µg/mL). Eugenol presented higher antioxidant activity than S. aromaticum essential oil, with an EC50 of 12.66 and 78.98 µg/mL, respectively. S. aromaticum essential oil and eugenol exhibited Trypanosoma cruzi inhibitory activity, with IC50 of 28.68 ± 1.073 and 31.97 ± 1.061 µg/mL against epimastigotes and IC50 of 64.51 ± 1.658 and 45.73 ± 1.252 µg/mL against intracellular amastigotes, respectively. Both compounds presented low cytotoxicity, with S. aromaticum essential oil displaying 15.5-fold greater selectivity for the parasite than the cells. Nitrite levels in T. cruzi-stimulated cells were reduced by essential oil (47.01%; p = 0.002) and eugenol (48.05%; p = 0.003) treatment. The trypanocidal activity of S. aromaticum essential oil showed that it is reasonable to use it in future research in the search for new therapeutic alternatives for trypanosomiasis.

Aniba rosaeodora (Var. amazonica Ducke) Essential Oil: Chemical Composition, Antibacterial, Antioxidant and Antitrypanosomal Activity.

Aniba rosaeodora is one of the most widely used plants in the perfumery industry, being used as medicinal plant in the Brazilian Amazon. This work aimed to evaluate the chemical composition of A. rosaeodora essential oil and its biological activities. A. rosaeodora essential oil presented linalool (93.60%) as its major compound. The A. rosaeodora essential oil and linalool showed activity against all the bacteria strains tested, standard strains and marine environment bacteria, with the lower minimum inhibitory concentration being observed for S. aureus. An efficient antioxidant activity of A. rosaeodora essential oil and linalool (EC50: 15.46 and 6.78 µg/mL, respectively) was evidenced by the inhibition of the 2,2-azinobis- (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical. The antitrypanosomal activity of A. rosaeodora essential oil and linalool was observed at high concentrations against epimatigote forms (inhibitory concentration for 50% of parasites (IC50): 150.5 ± 1.08 and 198.6 ± 1.12 µg/mL, respectively), and even higher against intracellular amastigotes of T. cruzi (IC50: 911.6 ± 1.15 and 249.6 ± 1.18 µg/mL, respectively). Both A. rosaeodora essential oil and linalool did not exhibit a cytotoxic effect in BALB/c peritoneal macrophages, and both reduced nitrite levels in unstimulated cells revealing a potential effect in NO production. These data revealed the pharmacological potential of A. rosaeodora essential oil and linalool, encouraging further studies.

Vernonia polysphaera Baker: Anti-inflammatory activity in vivo and inhibitory effect in LPS-stimulated RAW 264.7 cells.

Species of the Vernonia genius are widely distributed across the world. In traditional communities, they are commonly used in popular medicine for the treatment of inflammatory diseases. The objective of the present study was to evaluate the anti-inflammatory activity of Vernonia polysphaera Baker hydroalcoholic extract. A λ-carrageenan-induced paw edema and peritonitis model was established in BALB/c mice. The in vitro activity of the extract was measured on LPS-stimulated RAW 264.7 cells. There was no toxic effect on mice or on the cells treated with the extract. Animals treated with V. polysphaera extract demonstrated inhibition of paw edema in comparison with the untreated animals at all the analyzed doses. In peritonitis, treatment with the extract at a dose of 500 mg/kg resulted in a lower total leukocyte count in the peritoneal fluid and blood and lower levels of IL-1ß, IL-6, TNF-α and PGE-2 than the control group. Cells treated with 50 and 100 µg/mL of the extract exhibited lower levels of nitrite and pro-inflammatory cytokine production and lower COX-2, NF-κB expression. The V. polysphaera extract demonstrated an anti-inflammatory effect, interfering with cell migration, reducing pro-inflammatory cytokine levels and COX-2 expression and consequent interference with PGE-2, as well as inhibiting NF-κB transcription.

Endlicheria bracteolata (Meisn.) Essential Oil as a Weapon Against Leishmania amazonensis: In Vitro Assay.

The difficulties encountered and the numerous side effects present in the treatment of cutaneous leishmaniasis have encouraged the research for new compounds that can complement or replace existing treatment. The growing scientific interest in the study of plants, which are already used in folk remedies, has led our group to test Endlicheria bracteolata essential oil against Leishmania amazonensis. Several species of the Lauraceae family, or their compounds, have relevant antiprotozoal activities Therefore, the biological potential on L. amazonensis forms from the essential oil of Endlicheria bracteolata leaves was verified for the first time in that work. The antileishmanial activity was evaluated against promastigotes and intracellular amastigotes, and cytotoxicity were performed with J774.G8, which were incubated with different concentrations of E. bracteolata essential oil. Transmission electron microscopy and flow cytometry were performed with E. bracteolata essential oil IC50. Promastigote forms showed E. bracteolata essential oil IC50 of 7.945 ± 1.285 µg/mL (24 h) and 6.186 ± 1.226 µg/mL (48 h), while for intracellular amastigote forms it was 3.546 ± 1.184 µg/mL (24 h). The CC50 was 15.14 ± 0.090 µg/mL showing that E. bracteolata essential oil is less toxic to macrophages than to parasites. Transmission electron microscopy showed that E. bracteolata essential oil treatment is capable of inducing mitochondrial damage to promastigote and intracellular amastigote forms, while flow cytometry showed ΔÑ°m disruption in treated parasites. These results could bring about new possibilities to develop products based on E. bracteolata essential oil to treat cutaneous leishmaniasis, especially for people who cannot receive the conventional therapy.

Carapa guianensis Aublet (Andiroba) Seed Oil: Chemical Composition and Antileishmanial Activity of Limonoid-Rich Fractions.

Leishmaniasis is a complex of diseases caused by protozoa of the genus Leishmania and affects millions of people around the world. Several species of plants are used by traditional communities for the treatment of this disease, among which is Carapa guianensis Aubl. (Meliaceae), popularly known as andiroba. The objective of the present work was to conduct a chemical study of C. guianensis seed oil and its limonoid-rich fractions, with the aim of identifying its secondary metabolites, particularly the limonoids, in addition to investigating its anti-Leishmania potential. The chemical analyses of the C. guianensis seed oil and fractions were obtained by electrospray ionization mass spectrometry (ESI-MS). The cytotoxic activity was tested against peritoneal macrophages, and antileishmanial activity was evaluated against promastigotes and intracellular amastigotes of Leishmania amazonensis. All the C. guianensis seed oil samples analyzed exhibited the same pattern of fatty acids, while the limonoids 7-deacetoxy-7-hydroxygedunin, deacetyldihydrogedunin, deoxygedunin, andirobin, gedunin, 11ß-hydroxygedunin, 17-glycolyldeoxygedunin, 6α-acetoxygedunin, and 6α,11ß-diacetoxygedunin were identified in the limonoid-rich fractions of the oil. The C. guianensis seed oil did not exhibit antileishmanial activity, and cytotoxicity was higher than 1000 µg/mL. Three limonoid-rich oil fractions demonstrated activity against promastigotes (IC50 of 10.53±0.050, 25.3±0.057, and 56.9±0.043µg/mL) and intracellular amastigotes (IC50 of 27.31±0.091, 78.42±0.086, and 352.2±0.145 µg/mL) of L. amazonensis, as well as cytotoxicity against peritoneal macrophages (CC50 of 78.55±1.406, 139.0±1.523, and 607.7±1.217 µg/mL). The anti-Leishmania activity of the limonoid-rich fractions of C. guianensis can be attributed to the limonoids 11ß-hydroxygedunin and 6α,11ß-diacetoxygedunin detected in the chemical analysis.

N-acetyl-cysteine inhibits liver oxidative stress markers in BALB/c mice infected with Leishmania amazonensis.

BACKGROUND: Leishmaniasis is a parasitosis caused by several species of the genus Leishmania. These parasites present high resistance against oxidative stress generated by inflammatory cells. OBJECTIVES: To investigate oxidative stress and molecular inflammatory markers in BALB/c mice infected with L. amazonensis and the effect of antioxidant treatment on these parameters. METHODS: Four months after infection, oxidative and inflammatory parameters of liver, kidneys, spleen, heart and lungs from BALB/c mice were assessed. FINDINGS: In liver, L. amazonensis caused thiol oxidation and nitrotyrosine formation; SOD activity and SOD2 protein content were increased while SOD1 protein content decreased. The content of the cytokines IL-1ß, IL-6, TNF-α, and the receptor of advanced glycation endproducts (RAGE) increased in liver. Treatment with the antioxidant N-acetyl-cysteine (20 mg/kg b.w) for five days inhibited oxidative stress parameters. MAIN CONCLUSIONS: L. amazonensis induces significant alterations in the redox status of liver but not in other organs. Acute antioxidant treatment alleviates oxidative stress in liver, but it had no effect on pro-inflammatory markers. These results indicate that the pathobiology of leishmaniasis is not restricted to the cutaneous manifestations and open perspectives for the development of new therapeutic approaches to the disease, especially for liver function.

N-acetyl-cysteine inhibits liver oxidative stress markers in BALB/c mice infected with Leishmania amazonensis

BACKGROUND Leishmaniasis is a parasitosis caused by several species of the genus Leishmania. These parasites present high resistance against oxidative stress generated by inflammatory cells. OBJECTIVES To investigate oxidative stress and molecular inflammatory markers in BALB/c mice infected with L. amazonensis and the effect of antioxidant treatment on these parameters. METHODS Four months after infection, oxidative and inflammatory parameters of liver, kidneys, spleen, heart and lungs from BALB/c mice were assessed. FINDINGS In liver, L. amazonensis caused thiol oxidation and nitrotyrosine formation; SOD activity and SOD2 protein content were increased while SOD1 protein content decreased. The content of the cytokines IL-1β, IL-6, TNF-α, and the receptor of advanced glycation endproducts (RAGE) increased in liver. Treatment with the antioxidant N-acetyl-cysteine (20 mg/kg b.w) for five days inhibited oxidative stress parameters. MAIN CONCLUSIONS L. amazonensis induces significant alterations in the redox status of liver but not in other organs. Acute antioxidant treatment alleviates oxidative stress in liver, but it had no effect on pro-inflammatory markers. These results indicate that the pathobiology of leishmaniasis is not restricted to the cutaneous manifestations and open perspectives for the development of new therapeutic approaches to the disease, especially for liver function.

Morinda citrifolia Linn. fruit (Noni) juice induces an increase in NO production and death of Leishmania amazonensis amastigotes in peritoneal macrophages from BALB/c.

Leishmaniasis is a complex disease that is considered a serious public health problem. Due to the absence of an effective vaccine and debilitating chemotherapy better therapies are urgently needed. This situation has stimulated the search for alternative treatments such as the use of herbal medicines. Several studies conducted with Morinda citrifolia Linn. have shown various biological activities such as antitumor, immunomodulation and antileishmanial activity, however its mechanisms of action are still unknown. This study aimed to analyze the activity of M. citrifolia fruit juice against Leishmania amazonensis and its action on peritoneal macrophages from BALB/c infected with L. amazonensis. Activity against the promastigote forms showed IC50 at 275.3 µg/mL. Transmission electron microscopy was used to evaluate the ultrastructural alterations in the promastigotes treated with the juice and the results showed cytoplasmic vacuolization, lipid inclusion and increased activity of exocytosis. The juice treatment presented an IC50 at 208.4 µg/mL against intracellular amastigotes and led to an increased nitrite production in infected and non-infected macrophages. When macrophages were pre-treated with iNOS inhibitors, aminoguanidine or 1400W, the intracellular amastigotes increased, demonstrating the important role of NO production in M. citrifolia fruit activity. In conclusion, our results reveal that treatment with M. citrifolia fruit juice can increase NO production in peritoneal macrophages and this ability has an important role in the killing of L. amazonensis intracellular amastigotes.

In vitro evaluation of (-)α-bisabolol as a promising agent against Leishmania amazonensis.

Current treatments for leishmaniasis present some difficulties due to their toxicity, the use of the intravenous route for administration and therapy duration, which may lead to treatment discontinuation. The aim of this study is to investigate new treatment alternatives to improve patients well being. Therefore, we evaluated the inhibitory effect of (-)α-bisabolol, a sesquiterpene alcohol found in various essential oils of different plant species, against the promastigotes and intracellular amastigotes forms of Leishmania amazonensis, as well as the cytotoxic, morphological and ultrastructural alterations of treated cells. Promastigotes forms of L. amazonensis were incubated with (-)α-bisabolol to determine the antileishmanial activity of this compound. The cytotoxicity effect was evaluated by testing against J774.G8 cells. After these tests, the infected and uninfected cells with L. amazonensis were used to determine if the (-)α-bisabolol was able to kill intracellular parasites and to cause some morphological changes in the cells. The (-)α-bisabolol compound showed significant antileishmanial activity against promastigotes with a 50% effective concentration of 8.07 µg/ml (24 h) and 4.26 µg/ml (48 h). Against intracellular amastigotes the IC50 (inhibitory concentration) of (-)α-bisabolol (24 h) was 4.15 µg/ml. The (-)α-bisabolol also showed a cytotoxic effect against the macrophage strain J774.G8. The value of 50% cytotoxic concentration was 14.82 µg/ml showing that (-)α-bisabolol is less toxic to macrophages than to the parasite. Ultrastructural studies of treated promastigotes and amastigotes showed several alterations, such as loss of cytoplasmic organelles, including the nucleus, and the presence of lipid inclusions. This study showed that (-)α-bisabolol has promising antileishmanial properties, as it can act against the promastigote forms and is able to penetrate the cell, and is also active against the amastigote forms. About 69% of the promastigotes forms suffered mitochondrial membrane damage after treatment with IC50 of (-)α-bisabolol, suggesting inhibition of the metabolic activity of parasites. These results open new prospects for research that can contribute to the development of products based on essential oils or isolated compounds from plants for the treatment of cutaneous leishmaniasis.

Avaliação da capacidade leishmanicida de espécies vegetais do cerrado / Evaluation of the leishmanicidal activity of plant species of the Brazilian savanna

Para identificar as propriedades químicas de plantas do Cerrado, realizou-se um levantamentoetnobotânico que resultou na seleção de sete espécies vegetais de acordo com as indicações terapêuticaspara antibiótico, anti-inflamatório, analgésico, antiofídico e cicatrizante. Foram testados os extratoshidroalcoólicos das seguintes plantas quanto à sua atividade contra as formas promastigotas de Leishmaniaamazonensis: Terminalia fagifolia Mart. (Combretaceae), Vellozia squamata Pohl. (Velloziaceae),Vochysia haenkeana (Spreng.) Mart. (Vochysyaceae), Siparuna guianensis Aublet (Siparunaceae),Lafoensia pacari St. Hil. (Lythraceae), Galactia glauscecens Kunth. (Leguminosae) e Plathymeniareticulata Benth. (Mimosaceae). Feita a triagem para identificação da atividade leishmanicida,calculou-se a concentração inibitória do crescimento (IC50) em relação às culturas não tratadas com osextratos. Nas espécies L. pacari, G. glaucensces e P. reticulata, seus extratos demonstraram IC50 comvalores de 14,6 mug/mL, 46,0 mug/mL e 59,5 mug/mL, respectivamente, apresentando maior eficácia eminduzir a morte dos parasitos. T. fagifolia, V. squamata e V. haenkeana apresentaram IC50 com valoresde 446,1 mug/mL, 305,0 mug/mL e 85,1 mug/mL, respectivamente, demonstrando eficácia moderada.A prospecção fitoquímica evidenciou a presença de flavonoides, triterpenoides, esteroides e taninosque, segundo a literatura, são responsáveis pela atividade leishmanicida. Esses resultados indicam anecessidade de mais estudos para a avaliação da atividade em infecções in vivo e do fracionamento dassubstâncias em busca dos princípios ativos responsáveis pela ação leishmanicida...

In order to identify chemical properties of plants of the Brazilian savanna (Cerrado) an ethnobotanicalsurvey was performed. Seven plant species were selected according to their indications for antibiotic,anti-inflammatory, analgesic, anti-ophidian and healing properties. The following hydroalcoholic extracts of these plants were tested for activity against promastigotes of Leishmania amazonensis:Terminalia fagifolia Mart. (Combretaceae), Vellozia squamata Pohl. (Velloziaceae), Vochysiahaenkeana (Spreng.) Mart. (Vochysyaceae), Siparuna guianensis Aublet (Siparunaceae), Lafoensiapacari St. Hil. (Lythraceae), Galactia glauscecens Kunth. (Leguminosae) and Plathymeniareticulata Benth. (Mimosaceae). The growth inhibitory concentration (IC50) of these extracts wascalculated in relation to untreated cultures. Higher efficacy in inducing the death of parasites wasdemonstrated with extracts of L. pacari, G. glaucensces and P. reticulata, which showed IC50 valuesof 14.6 mg/mL, 46.0 mg/mL and 59.5 mg/mL, respectively. Moderate effectiveness was shown withT. fagifolia, V. squamata and V. haenkeana extracts, with values of 446.1, mg/mL 305.0 mg/mL and85.1 mg/mL respectively. Phytochemical studies showed the presence of flavonoids, triterpenoids,steroids and tannins that, according to the literature, are responsible for the leishmanicidal activity.These results indicate the need for future studies to evaluate the activity of these extracts againstinfections in vivo, as well as the fractionation of substances, in search of the active componentsresponsible for the leishmanicidal action...

Toxoplasma gondii oral infection induces intestinal inflammation and retinochoroiditis in mice genetically selected for immune oral tolerance resistance.

Toxoplasmosis is a worldwide disease with most of the infections originating through the oral route and generates various pathological manifestations, ranging from meningoencephalitis to retinochoroiditis and inflammatory bowel disease. Animal models for these pathologies are scarce and have limitations. We evaluated the outcome of Toxoplasma gondii oral infection with 50 or 100 cysts of the ME-49 strain in two lines of mice with extreme phenotypes of susceptibility (TS) or resistance (TR) to immune oral tolerance. Therefore, the aim of this study was to evaluate the behaviour of TS and TR mice, orally infected by T. gondii, and determine its value as a model for inflammatory diseases study. Mortality during the acute stage of the infection for TR was 50% for both dosages, while 10 and 40% of the TS died after infection with these respective dosages. In the chronic stage, the remaining TS succumbed while TR survived for 90 days. The TS displayed higher parasite load with lower intestinal inflammation and cellular proliferation, notwithstanding myocarditis, pneumonitis and meningoencephalitis. TR presented massive necrosis of villi and crypt, comparable to inflammatory bowel disease, with infiltration of lymphoid cells in the lamina propria of the intestines. Also, TR mice infected with 100 cysts presented intense cellular infiltrate within the photoreceptor layer of the eyes, changes in disposition and morphology of the retina cell layers and retinochoroiditis. During the infection, high levels of IL-6 were detected in the serum of TS mice and TR mice presented high amounts of IFN-γ and TNF-α. Both mice lineages developed different disease outcomes, but it is emphasized that TR and TS mice presented acute and chronic stages of the infection, demonstrating that the two lineages offer an attractive model for studying toxoplasmosis.

Morphometric changes in C57BL/6 mice retina infected by Toxoplasma gondii ME 49 strain.

This study evaluated the morphometric implications in C57BL/6 mouse retina infected by Toxoplasma gondii, ME 49 strain. Twenty C57BL/6 female mice were divided into group 1 (n=8, intraperitoneally infected with 30 cysts of T. gondii ME 49 strain) and group 2 (n=12 non-infected controls). The eyes were enucleated on the 60th day after infection, fixed and processed for light microscopy. Changes in retinal thickness and in the perimeter/area ratio (P/A) of the retinal layers were analyzed by digital morphometry. We considered that P/A was the measurement of retinal architecture distortion induced by toxoplasmosis. This study considered the ganglion cells and nerve fiber layers as a monolayer, thus six layers of retina were evaluated: photoreceptors (PRL), outer nuclear (ONL), outer plexiform (OPL), inner nuclear (INL), inner plexiform (IPL) and ganglion cells/nerve fiber monolayer (GNL). Histological analysis of infected mouse retina showed inflammatory infiltrate, necrosis, glial reaction and distortion of the retina architecture. It also presented increased thickness (167.8±24.9µm versus 121.1±15.4µm, in controls) and increased retinal thickness within the retinitis foci (187.7±16.6µm versus 147.9±12.2µm out of the retinitis foci). A statistically significant difference in P/A was observed between infected and uninfected mouse retinas. The same was observed in PRL, OPL, INL and GNL. Retinal morphometry may be used to demonstrate differences between infected and uninfected mouse retinas.

In Vitro Antileishmanial Activity of Essential Oil of Vanillosmopsis arborea (Asteraceae) Baker.

The search for new immunopharmacological chemical agents to treat various diseases caused by bacteria, fungi, and protozoa, such as leishmaniasis, for example, has led to the exploration of potential products from plant species and their main active ingredients. Antimonial drugs are the current treatment for leishmaniasis. These drugs cause major side effects and frequent discontinuation of treatment. In this study, we evaluated the in vitro leishmanicidal activity of essential oil of Vanillosmopsis arborea (VAEO) and its major compound α -bisabolol against Leishmania amazonensis. The essential oil and α -bisabolol showed activity against promastigotes (IC50 7.35 and 4.95 µ g/mL resp.) and intracellular amastigotes (IC50 12.58 and 10.70 µ g/mL, resp.). Neither product showed any cytotoxicity on treated macrophages. The ultrastructural analysis of promastigotes incubated with VAEO or α -bisabolol at 30 µ g/mL, showed morphological changes with the accumulation of vesicles electrodense lipid inclusions. The results give evidence that both VAEO and α -bisabolol have potential as new therapeutic agents against leishmaniasis.

Infection of retinal epithelial cells with L. amazonensis impacts in extracellular matrix proteins.

One of the manifestations of leishmaniases is eye injuries which main characteristics are the injury of the anterior chamber of the eye and the resistance to specific treatments. The retinal pigment epithelial (RPE) cells participate in pathogen-induced intraocular inflammatory processes. We investigated Leishmania amazonensis-RPE cells relationship and its impact in laminin and fibronectin production. Using RPE cell (ARPE-19), we demonstrated that L. amazonensis adhere to these cells in the first hour of infection, whereas parasite internalization was only observed after 6 h. Seventy-two hours after infection, vacuoles with parasites debris were observed intracellularly, and no parasite were observed intra- or extracellularly at the 96 h, suggesting that Leishmania can infect ARPE-19 cells although this cells are able to clear the infection. Fibronectin and laminin were associated with L. amazonensis-ARPE-19 interaction. Confocal analysis showed no substantial alterations in fibronectin presence in ARPE-19-infected or ARPE-19-noninfected cells, whereas laminin levels increased three times 10 h after L. amazonensis infection. After this time, laminin levels decreased in infected cells. These results suggest that L. amazonensis-ARPE-19 infection induces increased production of laminin in the beginning of infection which may facilitate parasite-host cell interactions.

Histopathological analysis of initial cellular response in TLR-2 deficient mice experimentally infected by Leishmania (L.) amazonensis.

Tegumentary leishmaniasis is an important public health problem in several countries. The capacity of the Leishmania species, at the initial moments of the infection, to invade and survive inside the host cells involves the interaction of surface molecules that are crucial in determining the evolution of the disease. Using C57BL/6 wild-type and TLR-2(-/-) mice infected with L. (L.) amazonensis, we demonstrated that TLR-2(-/-) mice presented eosinophilic granuloma in the ear dermis, different from C57BL/6 wild-type mice that presented a cellular profile characterized mainly by mononuclear cell infiltrates, besides neutrophils and eosinophils, during the two first week of infection. When the parasite load was evaluated, we found that the absence of TLR-2 lead to a significant reduction of the infection in deficient mice, when compared with C57BL/6 mice which were more susceptible to the infection. Using TLR-2 deficient mice, it was possible to show that the absence of this receptor determined the reduction of the parasite load and the recruitment of inflammatory cells during the two first weeks after L. (L.) amazonensis infection.

Leishmania (Leishmania) amazonensis: experimental cutaneous leishmaniasis associated with systemic amyloidosis in mice.

We infected Swiss and C57BL/6 female mice in the left hind footpad with 10(4)Leishmania (L.) amazonensis promastigotes in stationary phase. The macroscopic examination showed a nodular non-ulcerated lesion at the site of inoculation and hepatic and spleenic enlargement. Histopathologically, the primary lesion showed an extensive liquefactive necrosis and inflammatory infiltrate, mainly consisting of macrophages filled with amastigotes, and rare lymphocytes. The inflammatory reaction in liver, spleen and kidney showed amyloid deposits. Additionally, C57BL/6 had accentuated amyloidosis in both ovarian cortical and medullar region and inflammatory infiltrates in the pancreas and adrenal gland.