The beneficial effect of fluoxetine on behavioral and cognitive changes in chronic experimental Chagas disease unveils the role of serotonin fueling astrocyte infection by Trypanosoma cruzi.

BACKGROUND: In Chagas disease (CD), a neglected tropical disease caused by the parasite Trypanosoma cruzi, the development of mental disorders such as anxiety, depression, and memory loss may be underpinned by social, psychological, and biological stressors. Here, we investigated biological factors underlying behavioral changes in a preclinical model of CD. METHODOLOGY/PRINCIPAL FINDINGS: In T. cruzi-infected C57BL/6 mice, a kinetic study (5 to 150 days postinfection, dpi) using standardized methods revealed a sequential onset of behavioral changes: reduced innate compulsive behavior, followed by anxiety and depressive-like behavior, ending with progressive memory impairments. Hence, T. cruzi-infected mice were treated (120 to 150 dpi) with 10 mg/Kg/day of the selective serotonin reuptake inhibitor fluoxetine (Fx), an antidepressant that favors neuroplasticity. Fx therapy reversed the innate compulsive behavior loss, anxiety, and depressive-like behavior while preventing or reversing memory deficits. Biochemical, histological, and parasitological analyses of the brain tissue showed increased levels of the neurotransmitters GABA/glutamate and lipid peroxidation products and decreased expression of brain-derived neurotrophic factor in the absence of neuroinflammation at 150 dpi. Fx therapy ameliorated the neurochemical changes and reduced parasite load in the brain tissue. Next, using the human U-87 MG astroglioma cell line, we found no direct effect of Fx on parasite load. Crucially, serotonin/5-HT (Ser/5-HT) promoted parasite uptake, an effect increased by prior stimulation with IFNγ and TNF but abrogated by Fx. Also, Fx blocked the cytokine-driven Ser/5-HT-promoted increase of nitric oxide and glutamate levels in infected cells. CONCLUSION/SIGNIFICANCE: We bring the first evidence of a sequential onset of behavioral changes in T. cruzi-infected mice. Fx therapy improves behavioral and biological changes and parasite control in the brain tissue. Moreover, in the central nervous system, cytokine-driven Ser/5-HT consumption may favor parasite persistence, disrupting neurotransmitter balance and promoting a neurotoxic environment likely contributing to behavioral and cognitive disorders.

7-Aryl-7-deazapurine 3'-deoxyribonucleoside derivative as a novel lead for Chagas' disease therapy: in vitro and in vivo pharmacology.

BACKGROUND: The protozoan Trypanosoma cruzi is auxotrophic for purines and causes Chagas' disease (CD), a neglected illness affecting >6 million people. Combining the 3-deoxyribofuranose part of cordycepin with the modified purine ring of a nucleoside 'hit' led to the discovery of 4-amino-5-(4-chlorophenyl)-N7-(3'-deoxy-ß-d-ribofuranosyl)-pyrrolo[2,3-d]pyrimidine (Cpd1), revealing promising anti-T. cruzi activity. OBJECTIVES: To further evaluate Cpd1 in vitro and in vivo to fully assess its therapeutic potential against CD, covering cell culture sterilization through washout assays, drug combination with benznidazole and long-term administration in T. cruzi-infected mice. RESULTS: Although less susceptible to Cpd1 than amastigotes, trypomastigotes present an impaired capacity to successfully establish intracellular infection of cardiac cultures. Combination of benznidazole with Cpd1 indicated no interaction (additive effect) (FIC index = 0.72) while administration to mice at one-tenth of the optimal dose (2.5 mg/kg and 10 mg/kg for Cpd1 and benznidazole, respectively) suppressed parasitaemia but failed to avoid mortality. Long-term treatment (60 days) gave a rapid drop of the parasitaemia (>98% decline) and 100% mice survival but only 16% cure. In vitro washout experiments demonstrated that although parasite release into the supernatant of infected cardiac cultures was reduced by >94%, parasite recrudescence did occur after treatment. CONCLUSIONS: Parasite recrudescence did occur after treatment corroborating the hypothesis of therapeutic failure due to subpopulations of dormant forms and/or genetic factors in persister parasites involved in natural drug resistance.

6-Methyl-7-Aryl-7-Deazapurine Nucleosides as Anti-Trypanosoma cruzi Agents: Structure-Activity Relationship and in†vivo Efficacy.

Chagas disease is a tropical infectious disease resulting in progressive organ-damage and currently lacks efficient treatment and vaccine options. The causative pathogen, Trypanosoma cruzi, requires uptake and processing of preformed purines from the host because it cannot synthesize these de novo, instigating the evaluation of modified purine nucleosides as potential trypanocides. By modifying the pyrimidine part of a previously identified 7-aryl-7-deazapurine nucleoside, we found that substitution of a 6-methyl for a 6-amino group allows retaining T. cruzi amastigote growth inhibitory activity but confers improved selectivity towards mammalian cells. By keeping the 6-methyl group unaltered, and introducing different 7-aryl groups, we identified several analogues with sub-micromolar antitrypanosomal activity. The 7-(4-chlorophenyl) analogue 14, which was stable in microsomes, was evaluated in an acute mouse model. Oral administration of 25 mg/kg b.i.d. suppressed peak parasitemia and protected mice from infection-related mortality, gave similar reductions as the reference drug of blood parasite loads determined by qPCR, but as benznidazole failed to induce sterile cure in the short time period of drug exposure (5 days).

Memory impairment in chronic experimental Chagas disease: Benznidazole therapy reversed cognitive deficit in association with reduction of parasite load and oxidative stress in the nervous tissue.

Memory impairment has been associated with chronic Chagas disease (CD), a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. In degenerative diseases, memory loss has been associated with increased oxidative stress, revealed as enhanced lipid peroxidation, in the cerebral cortex. Benznidazole (Bz), a trypanocidal drug efficient to reduce blood parasite load in the acute and chronic phases of infection, showed controversial effects on heart disease progression, the main clinical manifestation of CD. Here, we evaluated whether C57BL/6 mice infected with the Colombian type I T. cruzi strain present memory deficit assessed by (i) the novel object recognition task, (ii) the open field test and (iii) the aversive shock evoked test, at 120 days post infection (dpi). Next, we tested the effects of Bz therapy (25mg/Kg/day, for 30 consecutive days) on memory evocation, and tried to establish a relation between memory loss, parasite load and oxidative stress in the central nervous system (CNS). At 120 dpi, T. cruzi-infected mice showed memory impairment, compared with age-matched non-infected controls. Bz therapy (from 120 to 150 dpi) hampered the progression of habituation and aversive memory loss and, moreover, reversed memory impairment in object recognition. In vehicle-administered infected mice, neuroinflammation was absent albeit rare perivascular mononuclear cells were found in meninges and choroid plexus. Bz therapy abrogated the infiltration of the CNS by inflammatory cells, and reduced parasite load in hippocampus and cerebral cortex. At 120 and 150 dpi, lipid peroxidation was increased in the hippocampus and cortex tissue extracts. Notably, Bz therapy reduced levels of lipid peroxidation in the cerebral cortex. Therefore, in experimental chronic T. cruzi infection Bz therapy improved memory loss, in association with reduction of parasite load and oxidative stress in the CNS, providing a new perspective to improve the quality of life of Chagas disease patients.

TGF-ß inhibitor therapy decreases fibrosis and stimulates cardiac improvement in a pre-clinical study of chronic Chagas' heart disease.

TGF-ß involvement in Chagas disease cardiomyopathy has been clearly demonstrated. The TGF-ß signaling pathway is activated in the cardiac tissue of chronic phase patients and is associated with an increase in extracellular matrix protein expression. The aim of this study was to investigate the effect of GW788388, a selective inhibitor of TßR1/ALK5, on cardiac function in an experimental model of chronic Chagas' heart disease. To this end, C57BL/6 mice were infected with Trypanosoma cruzi (102 parasites from the Colombian strain) and treated orally with 3mg/kg GW788388 starting at 120 days post-infection (dpi), when 100% of the infected mice show cardiac damage, and following three distinct treatment schedules: i) single dose; ii) one dose per week; or iii) three doses per week during 30 days. The treatment with GW788388 improved several cardiac parameters: reduced the prolonged PR and QTc intervals, increased heart rate, and reversed sinus arrhythmia, and atrial and atrioventricular conduction disorders. At 180 dpi, 30 days after treatment interruption, the GW3x-treated group remained in a better cardiac functional condition. Further, GW788388 treatment reversed the loss of connexin-43 enriched intercellular plaques and reduced fibrosis of the cardiac tissue. Inhibition of the TGF-ß signaling pathway reduced TGF-ß/pSmad2/3, increased MMP-9 and Sca-1, reduced TIMP-1/TIMP-2/TIMP-4, and partially restored GATA-6 and Tbox-5 transcription, supporting cardiac recovery. Moreover, GW788388 administration did not modify cardiac parasite load during the infection but reduced the migration of CD3+ cells to the heart tissue. Altogether, our data suggested that the single dose schedule was not as effective as the others and treatment three times per week during 30 days seems to be the most effective strategy. The therapeutic effects of GW788388 are promising and suggest a new possibility to treat cardiac fibrosis in the chronic phase of Chagas' heart disease by TGF-ß inhibitors.

Impact of levamisole in co-administration with benznidazole on experimental Chagas disease.

Levamisole (Lms) is an anthelminthic drug with immunomodulatory activity. Chagas disease (CD) is caused by Trypanosoma cruzi and there is very low access to the drugs available, benznidazole (Bz) and nifurtimox, both far from ideal. In a drug-repurposing strategy to test potential activity as antiparasitic and immunomodulatory agent for CD, Lms was assayed on acute T. cruzi murine infection, alone and in co-administration with Bz. During protocol standardization, 100 and 10 mpk of Bz given for five consecutive days resulted in parasitaemia suppression and 100% animal survival only with the highest dose. Flow cytometry showed that both optimal (100 mpk) and suboptimal (10 mpk) doses of Bz equally decreased the plasma levels of cytokines commonly elevated in this acute infection model. Lms alone (10-0.5 mpk) did not decrease parasitaemia nor mortality rates. Co-administration was investigated using the suboptimal dose of Bz and different doses of Lms. While Bz 10 mpk did not alter parasitaemia, the combo partially reduced it but only slightly promoted animal survival. This effect could be related to Th1-response modulation since interleukin-6 and interferon-γ were higher after treatment with the combo.

Assessing Parasite Load in Chagas Disease Patients by Quantitative Multiplex Real-Time PCR.

The development of accurate diagnostic tools and surrogate markers of parasitological response to treatment are priorities in Chagas disease (CD) research. For years, the detection of Trypanosoma cruzi DNA by PCR has proved to be useful in some clinical scenarios like acute CD, including cases of congenital transmission, CD reactivation in immunosuppressed patients, and posttreatment follow-up. In that sense, the implementation of quantitative real-time PCR (qPCR) assays was an important step in the development of more reliable tools for CD molecular diagnostics and treatment follow-up. In the last decade, two multicenter PCR studies allowed the harmonization and validation of standard operating procedures for PCR-based detection and quantification of T. cruzi DNA in blood samples. Herein we describe the two most used protocols to quantify parasitic load in human blood samples by multiplex qPCR assays and discuss some aspects to consider during planning and executing these procedures.

Genotyping of Trypanosoma cruzi from Clinical Samples by Multilocus Conventional PCR.

By the most recent nomenclature, Trypanosoma cruzi isolates are classified into six discrete typing units (DTUs)-T. cruzi I to T. cruzi VI and TcBat. One of the major challenges in the Chagas disease study is to find an association between DTUs and clinical manifestations of the disease or response to treatment. Herein, a protocol based on the amplification of T. cruzi SL-IRac, SL-IR I and II, 24Sα rDNA, and A10 targets by multilocus conventional PCRs is described. Following this methodology, it is possible to perform the genotyping directly from the blood and other clinical samples, without the need to isolate the parasite prior to the DNA extraction, even in a lower parasite concentration. Furthermore, this methodology increases the probability to detect mixed infections, avoiding a possible selection of strains during the parasite isolation.

Combination Chemotherapy with Suboptimal Doses of Benznidazole and Pentoxifylline Sustains Partial Reversion of Experimental Chagas' Heart Disease.

Chronic chagasic cardiomyopathy (CCC) progresses with parasite persistence, fibrosis, and electrical alterations associated with an unbalanced immune response such as high plasma levels of tumor necrosis factor (TNF) and nitric oxide (NO). Presently, the available treatments only mitigate the symptoms of CCC. To improve CCC prognosis, we interfered with the parasite load and unbalanced immune response using the trypanocidal drug benznidazole (Bz) and the immunoregulator pentoxifylline (PTX). C57BL/6 mice chronically infected with the Colombian strain of Trypanosoma cruzi and with signs of CCC were treated for 30 days with a suboptimal dose of Bz (25 mg/kg of body weight), PTX (20 mg/kg), or their combination (Bz plus PTX) and analyzed for electrocardiographic, histopathological, and immunological changes. Bz (76%) and Bz-plus-PTX (79%) therapies decreased parasite loads. Although the three therapies reduced myocarditis and fibrosis and ameliorated electrical alterations, only Bz plus PTX restored normal heart rate-corrected QT (QTc) intervals. Bz-plus-PTX-treated mice presented complementary effects of Bz and PTX, which reduced TNF expression (37%) in heart tissue and restored normal TNF receptor 1 expression on CD8(+) T cells, respectively. Bz (85%) and PTX (70%) therapies reduced the expression of inducible nitric oxide synthase (iNOS/NOS2) in heart tissue, but only Bz (58%) reduced NO levels in serum. These effects were more pronounced after Bz-plus-PTX therapy. Moreover, 30 to 50 days after treatment cessation, reductions of the prolonged QTc and QRS intervals were sustained in Bz-plus-PTX-treated mice. Our findings support the importance of interfering with the etiological agent and immunological abnormalities to improve CCC prognosis, opening an opportunity for a better quality of life for Chagas' disease (CD) patients.

Analytical Validation of Quantitative Real-Time PCR Methods for Quantification of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients.

An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease.

Selenium Treatment and Chagasic Cardiopathy (STCC): study protocol for a double-blind randomized controlled trial.

BACKGROUND: Heart disease progression occurs in 30% of patients with chronic Trypanosoma cruzi infection. Supplementation with selenium (Se) in animal model of T. cruzi infection produced promising results. There is evidence that patients with Chagas heart disease have lower Se levels than healthy individuals and patients with T. cruzi infection without of cardiac disease. The aim of this investigation is to estimate the effect of Se treatment on prevention of heart disease progression in patients with chagasic cardiopathy. METHODS: The Selenium Treatment and Chagasic Cardiopathy trial is a superiority, double-blind, placebo-controlled, randomized clinical trial. The eligibility criteria are as follows: (1) a Chagas disease diagnosis confirmed by serology; (2) segmental, mild or moderate global left ventricular systolic dysfunction; and (3) age between 18 and 65 years. The exclusion criteria are as follows: (1) pregnancy, (2) diabetes mellitus, (3) tobacco use, (4) alcohol abuse, (5) evidence of nonchagasic heart disease, (6) depression, (7) dysphagia with evidence of food residues in the esophagus, (8) dysphagia with weight loss higher than 15% of usual weight in the last four months and/or (9) conditions that may result in low protocol adherence. The intervention will be 100 µg of sodium selenite once daily for 365 consecutive days compared to placebo. The following are the primary outcomes to be measured: (1) the trajectories of the left ventricular ejection fraction in the follow-up period; (2) reduction of heart disease progression rates, with progression defined as a 10% decrease in left ventricular ejection fraction; and (3) rate of hospital admissions attributable to dysrhythmia, heart failure or stroke due to Chagas disease. One hundred thirty patients will be randomly allocated into either the intervention or placebo group at a ratio of 1:1. The sequence allocation concealment and blinding were planned to be conducted with the strategy of numbered boxes. Both patients and health-care providers will remain blinded to the intervention groups during the 5 years of follow-up. DISCUSSION: If Se treatment reduces the progression of Chagas cardiopathy, the inclusion of this micronutrient in the daily diet can improve the therapeutic regimen for this neglected tropical disease at low cost. TRIAL REGISTRATION: Clinical Trials.gov ID: NCT00875173 (registered 20 October 20 2008).