Intracerebral Injection of Heme Induces Lipid Peroxidation, Neuroinflammation, and Sensorimotor Deficits.

Background and Purpose: Heme is a red blood cell component released in the brain parenchyma following intracerebral hemorrhage. However, the study of the pathophysiological mechanisms triggered by heme in the brain is hampered by the lack of well-established in vivo models of intracerebral heme injection. This study aims to optimize and characterize a protocol of intrastriatal heme injection in mice, with a focus on the induction of lipid peroxidation, neuroinflammation and, ultimately, sensorimotor deficits. We also evaluated the involvement of NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3), an inflammasome sensor, in the behavior deficits induced by heme in this model. Methods: Mice were injected with heme in the striatum for the evaluation of neuroinflammation and brain damage through histological and biochemical techniques. Immunoblot was used to evaluate the expression of proteins involved in heme/iron metabolism and antioxidant responses and the activation of the MAPK (mitogen-activated protein kinase) signaling pathway. For the assessment of neurological function, we followed-up heme-injected mice for 2 weeks using the rotarod, elevated body swing, and cylinder tests. Mice injected with the vehicle (sham), or autologous blood were used as controls. Results: Heme induced lipid peroxidation and inflammation in the brain. Moreover, heme increased the expression of HO-1 (heme oxygenase-1), ferritin, p62, and superoxide dismutase 2, and activated the MAPK signaling pathway promoting pro-IL (interleukin)-1ß production and its cleavage to the active form. Heme-injected mice exhibited signs of brain damage and reactive astrogliosis around the injection site. Behavior deficits were observed after heme or autologous blood injection in comparison to sham-operated controls. In addition, behavior deficits and IL-1ß production were reduced in Nlrp3 knockout mice in comparison to wild-type mice. Conclusions: Our results show that intracerebral heme injection induces neuroinflammation, and neurological deficits, in an NLRP3-dependent manner, suggesting that this is a feasible model to evaluate the role of heme in neurological disorders.

Resveratrol Reverses Functional Chagas Heart Disease in Mice.

Chronic chagasic cardiomyopathy (CCC) develops years after acute infection by Trypanosoma cruzi and does not improve after trypanocidal therapy, despite reduction of parasite burden. During disease, the heart undergoes oxidative stress, a potential causative factor for arrhythmias and contractile dysfunction. Here we tested whether antioxidants/ cardioprotective drugs could improve cardiac function in established Chagas heart disease. We chose a model that resembles B1-B2 stage of human CCC, treated mice with resveratrol and performed electrocardiography and echocardiography studies. Resveratrol reduced the prolonged PR and QTc intervals, increased heart rates and reversed sinus arrhythmia, atrial and atrioventricular conduction disorders; restored a normal left ventricular ejection fraction, improved stroke volume and cardiac output. Resveratrol activated the AMPK-pathway and reduced both ROS production and heart parasite burden, without interfering with vascularization or myocarditis intensity. Resveratrol was even capable of improving heart function of infected mice when treatment was started late after infection, while trypanocidal drug benznidazole failed. We attempted to mimic resveratrol's actions using metformin (AMPK-activator) or tempol (SOD-mimetic). Metformin and tempol mimicked the beneficial effects of resveratrol on heart function and decreased lipid peroxidation, but did not alter parasite burden. These results indicate that AMPK activation and ROS neutralization are key strategies to induce tolerance to Chagas heart disease. Despite all tissue damage observed in established Chagas heart disease, we found that a physiological dysfunction can still be reversed by treatment with resveratrol, metformin and tempol, resulting in improved heart function and representing a starting point to develop innovative therapies in CCC.

Toxocara canis: Larvicidal activity of fatty acid amides.

Considering the therapeutic potential of fatty acid amides, the present study aimed to evaluate their in vitro activity against Toxocara canis larvae and their cytotoxicity for the first time. Linoleylpyrrolidilamide was the most potent, with a minimal larvicidal concentration (MLC) of 0.05 mg/mL and 27% cytotoxicity against murine peritoneal macrophages C57BL/6 mice, as assessed by the MTT assay.

Silymarin reduces profibrogenic cytokines and reverses hepatic fibrosis in chronic murine schistosomiasis.

In chronic schistosomiasis, hepatic fibrosis is linked to the portal hypertension that causes morbidity in Schistosoma mansoni infection. Silymarin (SIL) is a hepatoprotective and antioxidant medicament largely prescribed against liver diseases that has previously been shown to prevent fibrosis during acute murine schistosomiasis. Here we employed silymarin to try to reverse established hepatic fibrosis in chronic schistosomiasis. Silymarin or vehicle was administered to BALB/c mice every 48 h, starting on the 40th (80 days of treatment), 70th (50 days), or 110th (10 days) day postinfection (dpi). All mice were sacrificed and analyzed at 120 dpi. Treatment with silymarin reduced liver weight and granuloma sizes, reduced the increase in alanine aminotransferase and aspartate aminotransferase levels, and reduced the established hepatic fibrosis (assessed by hydroxyproline contents and picrosirius staining). Treatment with silymarin also reduced the levels of interleukin-13 (IL-13) in serum and increased the gamma interferon (IFN-γ)/IL-13 ratio. There was a linear correlation between IL-13 levels in serum and hydroxyproline hepatic content in both infected untreated and SIL-treated mice, with decreased IL-13 levels corresponding to decreased hydroxyproline hepatic contents. Treatment with either SIL or N-acetylcysteine reduced both proliferation of fibroblast cell lines and basal/IL-13-induced production of collagen I, indicating that besides inhibiting IL-13 production during infection, SIL antioxidant properties most likely contribute to inhibition of collagen production downstream of IL-13. These results show that silymarin interferes with fibrogenic cytokines, reduces established fibrosis, and inhibits downstream effects of IL-13 on fibrogenesis, indicating the drug as a safe and cheap treatment to liver fibrotic disease in schistosomiasis.

Preclinical evaluation of combined therapy with amiodarone and low-dose benznidazole in a mouse model of chronic Trypanosoma cruzi infection.

Chagasic chronic cardiomyopathy (CCC) is the primary clinical manifestation of Chagas disease (CD), caused by Trypanosoma cruzi. Current therapeutic options for CD are limited to benznidazole (Bz) and nifurtimox. Amiodarone (AMD) has emerged as most effective drug for treating the arrhythmic form of CCC. To address the effects of Bz and AMD we used a preclinical model of CCC. Female C57BL/6 mice were infected with T. cruzi and subjected to oral treatment for 30 consecutive days, either as monotherapy or in combination. AMD in monotherapy decreased the prolonged QTc interval, the incidence of atrioventricular conduction disorders and cardiac hypertrophy. However, AMD monotherapy did not impact parasitemia, parasite load, TNF concentration and production of reactive oxygen species (ROS) in cardiac tissue. Alike Bz therapy, the combination of Bz and AMD (Bz/AMD), improved cardiac electric abnormalities detected T. cruzi-infected mice such as decrease in heart rates, enlargement of PR and QTc intervals and increased incidence of atrioventricular block and sinus arrhythmia. Further, Bz/AMD therapy ameliorated the ventricular function and reduced parasite burden in the cardiac tissue and parasitemia to a degree comparable to Bz monotherapy. Importantly, Bz/AMD treatment efficiently reduced TNF concentration in the cardiac tissue and plasma and had beneficial effects on immunological abnormalities. Moreover, in the cardiac tissue Bz/AMD therapy reduced fibronectin and collagen deposition, mitochondrial damage and production of ROS, and improved sarcomeric and gap junction integrity. Our study underlines the potential of the Bz/AMD therapy, as we have shown that combination increased efficacy in the treatment of CCC.

IL-1ß is not critical to chronic heart dysfunction in mice with Chagas disease.

Long after Trypanosoma cruzi infection, 40% of individuals develop a progressive chronic chagasic cardiomyopathy (CCC), with systolic dysfunction and arrhythmias. Since we previously showed IL-1ß mediates the development of systolic dysfunction and cardiac arrhythmias in diabetes mellitus and cardiorenal syndrome, and IL-1ß remains elevated in Chagas disease patients, here we tested the role of IL-1ß in CCC using a mouse model. Mice deficient in IL-1R expression (Il-1r-/- ) survived acute T. cruzi infection with greater parasitemia than controls but did not lose weight as wild-type (WT) did. At the chronic stage, WT presented prolonged ventricular repolarization intervals (QJ), while Il-1r-/- presented intervals like noninfected controls. Infected Il-1r-/- and WT did not differ in stroke volume (SV), the incidence of cardiac arrhythmias on electrocardiography (EKG), whole heart action potential duration (APD), or the incidence of triggered activity after S1-S2 protocol, which is a measure of susceptibility to cardiac arrhythmias. We also treated chronically infected WT mice with an IL-1R antagonist, anakinra. Treatment shortened the QJ interval but did not improve the SV or the incidence of cardiac arrhythmias on EKG. Anakinra failed to reduce triggered activity following the electrical extra-stimulation protocol. In conclusion, the absence of functional IL-1ß/IL-1R signaling did not prevent or reverse the decrease of SV or the incidence of cardiac arrhythmias induced by chronic T. cruzi infection, implying this is not a critical mechanism in generating or maintaining CCC. Since similar cardiac abnormalities were previously credited to IL-1ß signaling, ruling out this mechanism is important to discourage further attempts of IL-1ß blockade as a therapeutical measure.

Silymarin treatment reduces granuloma and hepatic fibrosis in experimental schistosomiasis.

The schistosomiasis is a parasitic infection with relevant social impact and an important health problem in many countries around world. The pathology of this infection is characterized by a granulomatous reaction around parasite eggs and by hepatic fibrosis. Silymarin, a complex compound isolated from Silybum marianum (L.) Gaertner, have been described as hepatoprotective, antioxidant, antifibrotic, immunomodulator, and anti-neoplastic agent. Some of these capacities could potentially protect against pathology in schistosomiasis. Herein, we evaluated the effects of silymarin on parasite burden, granuloma sizes, and liver fibrosis, which are associated with severity and morbidity of this disease. BALB/c mice treated intraperitoneally with 10, 20, or 25 doses of silymarin (10 mg kg(-1)) suspended in carboxymethylcellulose were analyzed at 55 days post-infection. Silymarin (1) did not affect parasite oviposition capacity; (2) reduced granulomatous peri-ovular reaction in the liver, and (3) decreased hepatic fibrosis in this infection. Taken together, these data suggest that treatment with silymarin at acute phase of schistosomiasis may result in a mild course of murine schistosomiasis and can be a promising complementary treatment reverting sequelae of this infection.

Purinergic P2Y12 Receptor Activation in Eosinophils and the Schistosomal Host Response.

Identifying new target molecules through which eosinophils secrete their stored proteins may reveal new therapeutic approaches for the control of eosinophilic disorders such as host immune responses to parasites. We have recently reported the expression of the purinergic P2Y12 receptor (P2Y12R) in human eosinophils; however, its functional role in this cell type and its involvement in eosinophilic inflammation remain unknown. Here, we investigated functional roles of P2Y12R in isolated human eosinophils and in a murine model of eosinophilic inflammation induced by Schistosoma mansoni (S. mansoni) infection. We found that adenosine 5'-diphosphate (ADP) induced human eosinophils to secrete eosinophil peroxidase (EPO) in a P2Y12R dependent manner. However, ADP did not interfere with human eosinophil apoptosis or chemotaxis in vitro. In vivo, C57Bl/6 mice were infected with cercariae of the Belo Horizonte strain of S. mansoni. Analyses performed 55 days post infection revealed that P2Y12R blockade reduced the granulomatous hepatic area and the eosinophilic infiltrate, collagen deposition and IL-13/IL-4 production in the liver without affecting the parasite oviposition. As found for humans, murine eosinophils also express the P2Y12R. P2Y12R inhibition increased blood eosinophilia, whereas it decreased the bone marrow eosinophil count. Our results suggest that P2Y12R has an important role in eosinophil EPO secretion and in establishing the inflammatory response in the course of a S. mansoni infection.

ANTHELMINTIC ACTIVITY OF LAPACHOL, ß-LAPACHONE AND ITS DERIVATIVES AGAINST Toxocara canis LARVAE.

Anthelmintics used for intestinal helminthiasis treatment are generally effective; however, their effectiveness in tissue parasitosis (i.e. visceral toxocariasis) is moderate. The aim of this study was to evaluate the in vitro activity of lapachol, ß-lapachone and phenazines in relation to the viability of Toxocara canis larvae. A concentration of 2 mg/mL (in duplicate) of the compounds was tested using microculture plates containing Toxocara canis larvae in an RPMI-1640 environment, incubated at 37 °C in 5% CO2 tension for 48 hours. In the 2 mg/mL concentration, four phenazines, lapachol and three of its derivatives presented a larvicide/larvistatic activity of 100%. Then, the minimum larvicide/larvistatic concentration (MLC) test was conducted. The compounds that presented the best results were nor-lapachol (MLC, 1 mg/mL), lapachol (MLC 0.5 mg/mL), ß-lapachone, and ß-C-allyl-lawsone (MLC, 0.25 mg/mL). The larvae exposed to the compounds, at best MLC with 100% in vitro activity larvicide, were inoculated into healthy BALB/c mice and were not capable of causing infection, confirming the larvicide potential in vitro of these compounds.

ANTHELMINTIC ACTIVITY OF LAPACHOL, β-LAPACHONE AND ITS DERIVATIVES AGAINST Toxocara canis LARVAE / Atividade anti-helmíntica do lapachol, β-lapachona e derivados contra larvas de Toxocara canis

Anthelmintics used for intestinal helminthiasis treatment are generally effective; however, their effectiveness in tissue parasitosis (i.e. visceral toxocariasis) is moderate. The aim of this study was to evaluate the in vitro activity of lapachol, β-lapachone and phenazines in relation to the viability of Toxocara canis larvae. A concentration of 2 mg/mL (in duplicate) of the compounds was tested using microculture plates containing Toxocara canis larvae in an RPMI-1640 environment, incubated at 37 °C in 5% CO2 tension for 48 hours. In the 2 mg/mL concentration, four phenazines, lapachol and three of its derivatives presented a larvicide/larvistatic activity of 100%. Then, the minimum larvicide/larvistatic concentration (MLC) test was conducted. The compounds that presented the best results were nor-lapachol (MLC, 1 mg/mL), lapachol (MLC 0.5 mg/mL), β-lapachone, and β-C-allyl-lawsone (MLC, 0.25 mg/mL). The larvae exposed to the compounds, at best MLC with 100% in vitro activity larvicide, were inoculated into healthy BALB/c mice and were not capable of causing infection, confirming the larvicide potential in vitro of these compounds.

Os anti-helmínticos empregados no tratamento das helmintoses intestinais, de modo geral, são eficazes, porém nas parasitoses teciduais, como é o caso da toxocaríase visceral, a eficácia é moderada. Este estudo teve como objetivo avaliar in vitro a atividade do lapachol, β-lapachona e fenazinas derivadas da β-lapachona sobre a viabilidade de larvas de Toxocara canis. Os compostos foram testados na concentração de 2 mg/mL (em duplicata) em placas de microcultivo, contendo larvas de T. canis em meio RPMI-1640, sendo incubados, a 37 °C, em tensão de CO2 de 5%, por 48 horas. Na concentração de 2 mg/mL, quatro fenazinas, o lapachol e três derivados, apresentaram atividade larvicida/larvostática de 100%. A seguir, foi realizado o teste de concentração larvicida/larvostártica mínima (CLM). Os compostos que apresentaram os melhores resultados foram o nor-lapachol (CLM, 1 mg/mL), lapachol (CLM, 0,5 mg/mL), a β-lapachona e a β-C-alil-lausona (CLM, 0,25 mg/mL). As larvas expostas aos compostos, na melhor CLM 100% in vitro foram inoculadas em camundongos BALB/c saudáveis não sendo capazes de causar infecção, confirmando o potencial larvicida in vitro desses compostos.

Prevalência de enteroparasitas em crianças atendidas em unidades básicas de saúde em uma cidade do sul do Brasil / Prevalence of enteroparasites in children attended at basic health unities in a Brazilian southern city

As helmintoses e protozooses intestinais constituem sério problema de saúde pública nos países emdesenvolvimento. Este estudo analisou a prevalência de parasitas intestinais em crianças pré-escolaresatendidas em Unidades Básicas de Saúde (UBS) na cidade do Rio Grande, Rio Grande do Sul, Brasil.Foram examinadas amostras de fezes de 144 crianças, no período de outubro de 2009 a março de 2010.A prevalência de parasitas patogênicos foi de 17,4 %, em que foram detectados ovos dos nematoidesAscaris lumbricoides (11,1 %) e Trichuris trichiura (6,3 %), e cistos do protozoário Giardia lamblia (4,9 %).Foram também identificados cistos de protozoários considerados não patogênicos, como Entamoeba coli,Endolimax nana e Enteromonas hominis, em 28,5 % das crianças. Em relação às prevalências nas UBS, aspositividades foram de 36,4 % na UBS-1, 11,1 % na UBS-3, 14,8 % na UBS-4, 8 % na UBS-5 e 19,2 % naUBS-6. Na UBS-2 não foi detectada positividade nas amostras de fezes analisadas. A prevalência de parasitaspatogênicos registrada na população estudada sugere a ocorrência de prováveis condições de reinfecção e anecessidade de implementar medidas de saneamento básico e de programas que possibilitem a melhoria dacondição de vida dessas crianças.