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.

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.

Drug Repurposing in Chagas Disease: Chloroquine Potentiates Benznidazole Activity against Trypanosoma cruzi In Vitro and In Vivo.

Drug combinations and drug repurposing have emerged as promising strategies to develop novel treatments for infectious diseases, including Chagas disease. In this study, we aimed to investigate whether the repurposed drugs chloroquine (CQ) and colchicine (COL), known to inhibit Trypanosoma cruzi infection in host cells, could boost the anti-T. cruzi effect of the trypanocidal drug benznidazole (BZN), increasing its therapeutic efficacy while reducing the dose needed to eradicate the parasite. The combination of BZN and COL exhibited cytotoxicity to infected cells and low antiparasitic activity. Conversely, a combination of BZN and CQ significantly reduced T. cruzi infection in vitro, with no apparent cytotoxicity. This effect seemed to be consistent across different cell lines and against both the partially BZN-resistant Y and the highly BZN-resistant Colombiana strains. In vivo experiments in an acute murine model showed that the BZN+CQ combination was eight times more effective in reducing T. cruzi infection in the acute phase than BZN monotherapy. In summary, our results demonstrate that the concomitant administration of CQ and BZN potentiates the trypanocidal activity of BZN, leading to a reduction in the dose needed to achieve an effective response. In a translational context, it could represent a higher efficacy of treatment while also mitigating the adverse effects of high doses of BZN. Our study also reinforces the relevance of drug combination and repurposing approaches in the field of Chagas disease drug discovery.

Sulfadiazine Plus Pyrimethamine Therapy Reversed Multiple Behavioral and Neurocognitive Changes in Long-Term Chronic Toxoplasmosis by Reducing Brain Cyst Load and Inflammation-Related Alterations.

Toxoplasma gondii infects one-third of the world population. For decades, it has been considered a silent lifelong infection. However, chronically T. gondii-infected persons may present psychiatric and neurocognitive changes as anxiety, depression, and memory loss. In a model of long-term chronic infection, behavioral alterations parallel neuroinflammation and systemic high cytokine levels, and may reflect brain cyst load. Recent findings support that in chronic infection an active parasite-host interplay involves an immune-mediated control of tissue cysts. Here, we tested the idea that etiological treatment in chronic phase may add advantage to intrinsic immune-mediated cyst control and impact behavioral changes. Thus, we combined sulfadiazine-plus-pyrimethamine (S+P), the first-choice therapy for toxoplasmosis, to study the association of brain cyst load and biological processes related to the immune response (neuroinflammation, blood-brain barrier -BBB- disruption and serum cytokine levels), with behavioral and neurocognitive changes of long-term chronic infection. Female C57BL/6 mice (H-2b) were infected (5 cysts, ME-49 strain) and treated with S+P from 30 to 60 days postinfection (dpi), compared with vehicle (Veh)-treated and noninfected controls. At endpoints (pre-therapy, 30 dpi; S+P therapy, 60 dpi; after ceased therapy, 90 dpi), independent groups were subjected to behavioral tests, and brain tissues and sera were collected. Multiple behavioral and neurocognitive changes were detected in the early (30 dpi) and long-term (60 and 90 dpi) chronic infection. S+P therapy resolved locomotor alterations, anxiety, and depressive-like behavior, partially or transiently ameliorated hyperactivity and habituation memory loss. Analysis after therapy cessation showed that S+P therapy reduced the number of stimuli required for aversive memory consolidation. S+P therapy resulted in reduced brain cyst load, neuroinflammation and BBB disruption, and lowered systemic Th1-cytokine levels. Correlation analysis revealed association between IFNγ, TNF and MCP-1/CCL2 serum levels, brain cyst load and behavioral and neurocognitive alterations. Moreover, principal-component analysis (PCA-2D and 3D projections) highlighted distinction between clusters (noninfected; Veh-treated and S+P-treated infected). Thus, our data suggest that S+P therapy added gain to intrinsic brain cyst control and, direct or indirectly, ameliorated inflammation-related alterations, traits associated with behavioral and neurocognitive alterations.

Modulation of miR-145-5p and miR-146b-5p levels is linked to reduced parasite load in H9C2 Trypanosoma cruzi infected cardiomyoblasts.

In the heart tissue of acutely Trypanosoma cruzi-infected mice miR-145-5p and miR-146b-5p are, respectively, downregulated and upregulated. Here, we used the H9C2 rat cardiomyoblast cell line infected with the Colombian T. cruzi strain to investigate the parasite-host cell interplay, focusing on the regulation of miR-145-5p and miR-146b-5p expression. Next, we explored the effects of interventions with the trypanosomicidal drug Benznidazole (Bz) alone or combined with Pentoxifylline (PTX), a methylxanthine derivative shown to modulate immunological and cardiac abnormalities in a model of chronic chagasic cardiomyopathy, on parasite load and expression of miR-145-5p and miR-146b-5p. The infection of H9C2 cells with trypomastigote forms allowed parasite cycle with intracellular forms multiplication and trypomastigote release. After 48 and 144 h of infection, upregulation of miR-145-5p (24 h: 2.38 ± 0.26; 48 h: 3.15 ± 0.9-fold change) and miR-146b-5b (24 h: 2.60 ± 0.46; 48 h: 2.97 ± 0.23-fold change) was detected. The peak of both miRNA levels paralleled with release of trypomastigote forms. Addition of 3 µM and 10 µM of Bz 48 h after infection reduced parasite load but did not interfere with miR-145-5p and miR-146b-5p levels. Addition of PTX did not interfere with Bz-induced parasite control efficacy. Conversely, combined Bz + PTX treatment decreased the levels of both microRNAs, resembling the expression levels detected in non-infected H9C2 cells. Moreover, the use of miR-145-5p and miR-146b-5p mimic/inhibitor systems before infection of H9C2 cells decreased parasite load, 72 h postinfection. When H9C2 cells were treated with miR-145-5p and miR-146b-5p mimic/inhibitor 48 h after infection, all the used systems, except the miR-146b-5p inhibitor, reduced parasite load. Altogether, our data indicate that these microRNAs putatively control signaling pathways crucial for parasite-host cell interaction. Thus, miR-145-5p and miR-146b-5p deserve to be further investigated as biomarkers of parasite control and tools to identify therapeutic adjuvants to etiological treatment in Chagas disease.

Physical Exercise Promotes a Reduction in Cardiac Fibrosis in the Chronic Indeterminate Form of Experimental Chagas Disease.

Chagas disease (CD), caused by the protozoan Trypanosoma cruzi, is a neglected tropical disease and a health problem in Latin America. Etiological treatment has limited effectiveness in chronic CD; thus, new therapeutic strategies are required. The practice of physical exercises has been widely advocated to improve the quality of life of CD patients. The most frequent clinical CD manifestation is the chronic indeterminate form (CIF), and the effect of physical exercises on disease progression remains unknown. Here, in a CIF model, we aimed to evaluate the effect of physical exercises on cardiac histological, parasitological, mitochondrial, and oxidative metabolism, electro and echocardiographic profiles, and immunological features. To establish a CIF model, BALB/c and C57BL/6 mice were infected with 100 and 500 trypomastigotes of the Y T. cruzi strain. At 120 days postinfection (dpi), all mouse groups showed normal PR and corrected QT intervals and QRS complexes. Compared to BALB/c mice, C57BL/6 mice showed a lower parasitemia peak, mortality rate, and less intense myocarditis. Thus, C57BL/6 mice infected with 500 parasites were used for subsequent analyses. At 120 dpi, a decrease in cardiac mitochondrial oxygen consumption and an increase in reactive oxygen species (ROS) were detected. When we increased the number of analyzed mice, a reduced heart rate and slightly prolonged corrected QT intervals were detected, at 120 and 150 dpi, which were then normalized at 180 dpi, thus characterizing the CIF. Y-infected mice were subjected to an exercise program on a treadmill for 4 weeks (from 150 to 180 dpi), five times per week in a 30-60-min daily training session. At 180 dpi, no alterations were detected in cardiac mitochondrial and oxidative metabolism, which were not affected by physical exercises, although ROS production increased. At 120 and 180 dpi, comparing infected and non-infected mice, no differences were observed in the levels of plasma cytokines, indicating that a crucial biomarker of the systemic inflammatory profile was absent and not affected by exercise. Compared with sedentary mice, trained Y-infected mice showed similar parasite loads and inflammatory cells but reduced cardiac fibrosis. Therefore, our data show that physical exercises promote beneficial changes that may prevent CD progression.

Behavioral alterations in long-term Toxoplasma gondii infection of C57BL/6 mice are associated with neuroinflammation and disruption of the blood brain barrier.

The Apicomplexa protozoan Toxoplasma gondii is a mandatory intracellular parasite and the causative agent of toxoplasmosis. This illness is of medical importance due to its high prevalence worldwide and may cause neurological alterations in immunocompromised persons. In chronically infected immunocompetent individuals, this parasite forms tissue cysts mainly in the brain. In addition, T. gondii infection has been related to mental illnesses such as schizophrenia, bipolar disorder, depression, obsessive-compulsive disorder, as well as mood, personality, and other behavioral changes. In the present study, we evaluated the kinetics of behavioral alterations in a model of chronic infection, assessing anxiety, depression and exploratory behavior, and their relationship with neuroinflammation and parasite cysts in brain tissue areas, blood-brain-barrier (BBB) integrity, and cytokine status in the brain and serum. Adult female C57BL/6 mice were infected by gavage with 5 cysts of the ME-49 type II T. gondii strain, and analyzed as independent groups at 30, 60 and 90 days postinfection (dpi). Anxiety, depressive-like behavior, and hyperactivity were detected in the early (30 dpi) and long-term (60 and 90 dpi) chronic T. gondii infection, in a direct association with the presence of parasite cysts and neuroinflammation, independently of the brain tissue areas, and linked to BBB disruption. These behavioral alterations paralleled the upregulation of expression of tumor necrosis factor (TNF) and CC-chemokines (CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1ß and CCL5/RANTES) in the brain tissue. In addition, increased levels of interferon-gamma (IFNγ), TNF and CCL2/MCP-1 were detected in the peripheral blood, at 30 and 60 dpi. Our data suggest that the persistence of parasite cysts induces sustained neuroinflammation, and BBB disruption, thus allowing leakage of cytokines of circulating plasma into the brain tissue. Therefore, all these factors may contribute to behavioral changes (anxiety, depressive-like behavior, and hyperactivity) in chronic T. gondii infection.

"Chagas Express XXI": A new ArtScience social technology for health and science education-A case study in Brazilian endemic areas of Chagas disease with an active search of chronic cases.

BACKGROUND: Chagas Disease (CD) affects 6-7 million people worldwide and is related to poverty-promoting conditions. Chronic asymptomatic cases are mostly invisible to health systems. Aiming (1) to translate CD discoveries into education/information practices to raise alertness and empowerment of affected people; and (2) to perform an active search of CD cases, articulating intersectoral actions to improve the access of infected people to the local health service for the treatment of CD; our research group developed and tested under field conditions as innovative social technology: an itinerant education interdisciplinary setting named "Chagas Express XXI" (CE21). METHODOLOGY: CE21 was created as an "imaginary train" with ~40 ArtScience workshops, games, laboratory activities and conversation circles. An entry/exit plus six activity modules combined associations of affected people, microscopic observations, One Health education, and wellness activities. CE21 was conceived as a social technology, since all the processes were co-created with CD patients and inter-sector local partners. Descriptive statistics showed quantitative data collected throughout the expeditions (CD knowledge, serological results). Qualitative data accessed the public perceptions about the education activities. PRINCIPAL FINDINGS: CE21 was exhibited in local educational institutions (schools, universities) in four cities, engaging 2,117 people that evaluated the 41 activities carried out. Citizens and health professionals enjoyed acquisition of information related to blood, parasites, vectors, reservoirs, environmental changes, and social determinants of CD. Further, local legacies of 600 participants volunteer for health promotion groups and CD associations, local empowerment groups to fight for better health conditions, and 05 mural paintings. We observed that 81% of the participants ignored the possibility of treating CD while 52% of the participants requested a blood test for CD showing seropositivity in 20% of them. CONCLUSIONS: CE21 is a social technology potentially useful for health and science education and active search of asymptomatic CD chronic cases. Moreover, this technology may be adapted to understand and to cooperate in other potentially epidemic situations, especially NTDs related.

CCL3/Macrophage Inflammatory Protein-1α Is Dually Involved in Parasite Persistence and Induction of a TNF- and IFNγ-Enriched Inflammatory Milieu in Trypanosoma cruzi-Induced Chronic Cardiomyopathy.

CCL3, a member of the CC-chemokine family, has been associated with macrophage recruitment to heart tissue and parasite control in the acute infection of mouse with Trypanosoma cruzi, the causative agent of Chagas disease. Here, we approached the participation of CCL3 in chronic chagasic cardiomyopathy (CCC), the main clinical form of Chagas disease. We induced CCC in C57BL/6 (ccl3+/+) and CCL3-deficient (ccl3-/-) mice by infection with the Colombian Type I strain. In ccl3+/+ mice, high levels of CCL3 mRNA and protein were detected in the heart tissue during the acute and chronic infection. Survival was not affected by CCL3 deficiency. In comparison with ccl3+/+, chronically infected ccl3-/- mice presented reduced cardiac parasitism and inflammation due to CD8+ cells and macrophages. Leukocytosis was decreased in infected ccl3-/- mice, paralleling the accumulation of CD8+ T cells devoid of activated CCR5+ LFA-1+ cells in the spleen. Further, T. cruzi-infected ccl3-/-mice presented reduced frequency of interferon-gamma (IFNγ)+ cells and numbers of parasite-specific IFNγ-producing cells, while the T. cruzi antigen-specific cytotoxic activity was increased. Stimulation of CCL3-deficient macrophages with IFNγ improved parasite control, in a milieu with reduced nitric oxide (NOx) and tumor necrosis factor (TNF), but similar interleukin-10 (IL-10), concentrations. In comparison with chronically T. cruzi-infected ccl3+/+ counterparts, ccl3-/- mice did not show enlarged heart, loss of left ventricular ejection fraction, QTc prolongation and elevated CK-MB activity. Compared with ccl3+/+, infected ccl3-/- mice showed reduced concentrations of TNF, while IL-10 levels were not affected, in the heart milieu. In spleen of ccl3+/+ NI controls, most of the CD8+ T-cells expressing the CCL3 receptors CCR1 or CCR5 were IL-10+, while in infected mice these cells were mainly TNF+. Lastly, selective blockage of CCR1/CCR5 (Met-RANTES therapy) in chronically infected ccl3+/+ mice reversed pivotal electrical abnormalities (bradycardia, prolonged PR, and QTc interval), in correlation with reduced TNF and, mainly, CCL3 levels in the heart tissue. Therefore, in the chronic T. cruzi infection CCL3 takes part in parasite persistence and contributes to form a CD8+ T-cell and macrophage-enriched cardiac inflammation. Further, increased levels of CCL3 create a scenario with abundant IFNγ and TNF, associated with cardiomyocyte injury, heart dysfunction and QTc prolongation, biomarkers of severity of Chagas' heart disease.

Genetic Polymorphism at CCL5 Is Associated With Protection in Chagas' Heart Disease: Antagonistic Participation of CCR1+ and CCR5+ Cells in Chronic Chagasic Cardiomyopathy.

Chronic cardiomyopathy is the main clinical manifestation of Chagas disease (CD), a disease caused by Trypanosoma cruzi infection. A hallmark of chronic chagasic cardiomyopathy (CCC) is a fibrogenic inflammation mainly composed of CD8+ and CD4+ T cells and macrophages. CC-chemokine ligands and receptors have been proposed to drive cell migration toward the heart tissue of CD patients. Single nucleotide polymorphisms (SNPs) in CC-chemokine ligand and receptor genes may determine protein expression. Herein, we evaluated the association of SNPs in the CC-chemokines CCL2 (rs1024611) and CCL5 (rs2107538, rs2280788) and the CCL5/RANTES receptors CCR1 (rs3181077, rs1491961, rs3136672) and CCR5 (rs1799987) with risk and progression toward CCC. We performed a cross-sectional association study of 406 seropositive patients from endemic areas for CD in the State of Pernambuco, Northeast Brazil. The patients were classified as non-cardiopathic (A, n = 110) or cardiopathic (mild, B1, n = 163; severe, C, n = 133). Serum levels of CCL5 and CCL2/MCP-1 were elevated in CD patients but were neither associated with risk/severity of CCC nor with SNP genotypes. After logistic regression analysis with adjustment for the covariates gender and ethnicity, CCL5 -403 (rs2107538) CT heterozygotes (OR = 0.5, P-value = 0.04) and T carriers (OR = 0.5, P-value = 0.01) were associated with protection against CCC. To gain insight into the participation of the CCL5-CCR5/CCR1 axis in CCC, mice were infected with the Colombian T. cruzi strain. Increased CCL5 concentrations were detected in cardiac tissue. In spleen, frequencies of CCR1+ CD8+ T cells and CD14+ macrophages were decreased, while frequencies of CCR5+ cells were increased. Importantly, CCR1+CD14+ macrophages were mainly IL-10+, while CCR5+ cells were mostly TNF+. CCR5-deficient infected mice presented reduced TNF concentrations and injury in heart tissue. Selective blockade of CCR1 (Met-RANTES therapy) in infected Ccr5-/- mice supported a protective role for CCR1 in CCC. Furthermore, parasite antigen stimulation of CD patient blood cells increased the frequency of CCR1+CD8+ T cells and CCL5 production. Collectively, our data support that a genetic variant of CCL5 and CCR1+ cells confer protection against Chagas heart disease, identifying the CCL5-CCR1 axis as a target for immunostimulation.

Priming astrocytes with TNF enhances their susceptibility to Trypanosoma cruzi infection and creates a self-sustaining inflammatory milieu.

BACKGROUND: In conditions of immunosuppression, the central nervous sty 5ystem (CNS) is the main target tissue for the reactivation of infection by Trypanosoma cruzi, the causative agent of Chagas disease. In experimental T. cruzi infection, interferon gamma (IFNγ)+ microglial cells surround astrocytes harboring amastigote parasites. In vitro, IFNγ fuels astrocyte infection by T. cruzi, and IFNγ-stimulated infected astrocytes are implicated as potential sources of tumor necrosis factor (TNF). Pro-inflammatory cytokines trigger behavioral alterations. In T. cruzi-infected mice, administration of anti-TNF antibody hampers depressive-like behavior. Herein, we investigated the effects of TNF on astrocyte susceptibility to T. cruzi infection and the regulation of cytokine production. METHODS: Primary astrocyte cultures of neonatal C57BL/6 and C3H/He mice and the human U-87 MG astrocyte lineage were infected with the Colombian T. cruzi strain. Cytokine production, particularly TNF, and TNF receptor 1 (TNFR1/p55) expression were analyzed. Recombinant cytokines (rIFNγ and rTNF), the anti-TNF antibody infliximab, and the TNFR1 modulator pentoxifylline were used to assess the in vitro effects of TNF on astrocyte susceptibility to T. cruzi infection. To investigate the role of TNF on CNS colonization by T. cruzi, infected mice were submitted to anti-TNF therapy. RESULTS: rTNF priming of mouse and human astrocytes enhanced parasite/astrocyte interaction (i.e., the percentage of astrocytes invaded by trypomastigote parasites and the number of intracellular parasite forms/astrocyte). Furthermore, T. cruzi infection drove astrocytes to a pro-inflammatory profile with TNF and interleukin-6 production, which was amplified by rTNF treatment. Adding rTNF prior to infection fueled parasite growth and trypomastigote egression, in parallel with increased TNFR1 expression. Importantly, pentoxifylline inhibited the TNF-induced increase in astrocyte susceptibility to T. cruzi invasion. In T. cruzi-infected mice, anti-TNF therapy reduced the number of amastigote nests in the brain. CONCLUSIONS: Our data implicate TNF as a promoter of T. cruzi invasion of mouse and human astrocytes. Moreover, the TNF-enriched inflammatory milieu and enhanced TNFR1 expression may favor TNF signaling, astrocyte colonization by T. cruzi and egression of trypomastigotes. Therefore, in T. cruzi infection, a self-sustaining TNF-induced inflammatory circuit may perpetuate the parasite cycle in the CNS and ultimately promote cytokine-driven behavioral alterations.

Pentoxifylline reverses chronic experimental Chagasic cardiomyopathy in association with repositioning of abnormal CD8+ T-cell response.

BACKGROUND: Chronic chagasic cardiomyopathy (CCC), the main clinical sign of Chagas disease, is associated with systemic CD8+ T-cell abnormalities and CD8-enriched myocarditis occurring in an inflammatory milieu. Pentoxifylline (PTX), a phosphodiesterase inhibitor, has immunoregulatory and cardioprotective properties. Here, we tested PTX effects on CD8+ T-cell abnormalities and cardiac alterations using a model of experimental Chagas' heart disease. METHODOLOGY/PRINCIPAL FINDINGS: C57BL/6 mice chronically infected by the Colombian Trypanosoma cruzi strain and presenting signs of CCC were treated with PTX. The downmodulation of T-cell receptors on CD8+ cells induced by T. cruzi infection was rescued by PTX therapy. Also, PTX reduced the frequency of CD8+ T-cells expressing activation and migration markers in the spleen and the activation of blood vessel endothelial cells and the intensity of inflammation in the heart tissue. Although preserved interferon-gamma production systemically and in the cardiac tissue, PTX therapy reduced the number of perforin+ cells invading this tissue. PTX did not alter parasite load, but hampered the progression of heart injury, improving connexin 43 expression and decreasing fibronectin overdeposition. Further, PTX reversed electrical abnormalities as bradycardia and prolonged PR, QTc and QRS intervals in chronically infected mice. Moreover, PTX therapy improved heart remodeling since reduced left ventricular (LV) hypertrophy and restored the decreased LV ejection fraction. CONCLUSIONS/SIGNIFICANCE: PTX therapy ameliorates critical aspects of CCC and repositioned CD8+ T-cell response towards homeostasis, reinforcing that immunological abnormalities are crucially linked, as cause or effect, to CCC. Therefore, PTX emerges as a candidate to treat the non-beneficial immune deregulation associated with chronic Chagas' heart disease and to improve prognosis.

CCL2/MCP-1 controls parasite burden, cell infiltration, and mononuclear activation during acute Trypanosoma cruzi infection.

CCL2/MCP-1 has emerged recently as a critical factor in infectious and autoimmune myocarditis. In fact, this chemokine is produced in great amounts in hearts from Trypanosoma cruzi-infected mice and is known to enhance parasite uptake and destruction by macrophages. Herein, we studied the involvement of CCL2 in tissue inflammation and resistance to T. cruzi. Infected CCL2(-/-) mice developed higher parasitemias and died earlier than WT mice. Close to their death, T. cruzi-infected CCL2(-/-) presented greater amounts of TNF, IFN-gamma, and IL-10 in plasma than WTs and clinical signs of systemic inflammatory response. Amastigote nests were more frequent in hearts and livers from infected CCL2(-/-) tissues than in WTs, and reduced numbers of leukocytes infiltrated their tissues. Leukocytes formed diffuse but not focal infiltrates in hearts from infected CCL2(-/-) mice, and perivascular cuffs could still be found in their livers. Infected CCL2(-/-) mice had smaller percentages of activated CD11b (Mac-1)+CD107b (Mac-3)+ macrophages and CD8+CD69(hi) cells among heart and liver infiltrates than WTs (flow cytometry), indicating that CCL2 controls subset migration/activation. CCL2 accumulated among focal heart infiltrates, suggesting that this chemokine is involved in retention of mononuclear cells in particular spots. Peritoneal macrophages from CCL2(-/-) mice displayed decreased trypanocidal activity. Our results demonstrate that CCL2 contributes to reduce parasite growth and indicate that it does so by controlling the distribution, cellular composition, and state of activation of inflammatory infiltrates in acute T. cruzi infection.

Unraveling the lethal synergism between Trypanosoma cruzi infection and LPS: a role for increased macrophage reactivity.

Various infections sensitize to lethal shock by promoting hyperactivation of macrophages to LPS stimulation. Although macrophages are thought to be deactivated upon contact with apoptotic cells during Trypanosoma cruzi infection, T. cruzi infection also sensitizes mice to endotoxemia. Herein, we studied the mechanisms of sensitization to endotoxemia in T. cruzi-infected mice in order to solve the paradox. Live (but not fixed) trypomastigotes from various stocks sensitized mice to endotoxemia. Mice deficient in glycolipid recognition (TLR2(-/-) and CD1d(-/-)) were sensitized by infection to challenge with LPS. Infected mice hyperproduced TNF and IL-10 upon LPS challenge. Infected TNF-R1(-/-), macrophage migration inhibitory factor (MIF)(-/-) and IFN-gamma(-/-) mice were lethally sensitized, but infected TNF-R1(-/-) mice administered anti-MIF survived shock with LPS. Macrophages from infected mice hyperproduced TNF in response to LPS stimulation and displayed increased expression of TLR4 compared to non-infected controls. Treatment with the PGE(2) synthesis inhibitor acetylsalicylic acid (AAS) in vivo reduced parasitemia and enhanced LPS-stimulated production of TNF by macrophages, but the effect was less in infected mice than in normal mice. Nevertheless, AAS treatment did not increase the susceptibility of infected mice to sublethal shock with LPS. Our results point to independent MIF and TNF/TNF-R1 lethal pathways and suggest a role for hyperactivated macrophages in T. cruzi-sensitized LPS-induced shock.

Effects of perillyl alcohol in glial C6 cell line in vitro and anti-metastatic activity in chorioallantoic membrane model.

The search for new chemotherapeutic drugs has increased, especially for those that have a natural origin. The monoterpene perillyl alcohol (POH) has been shown to exert chemopreventive activity in mammary, liver, and lung tumor models. It has also been used to treat a variety of rodent cancers, including pancreatic and breast carcinomas. In vitro data suggest that it may be effective against neuroblastomas and leukemias. This work evaluates the effects of the treatment of murine glial C6 cells with perillyl alcohol. In vitro, our studies have indicated that POH inhibits proliferation of the C6 glial cell line. POH was logarithmically diluted in concentrations of 30% through 0.0003% and showed inhibition cell proliferation of 78.36% in concentration of 30%; 69.87% in concentration of 3%; and 67.04% in concentration of 0.03%. In addition, the anti-metastatic activity of POH against these cells was evaluated using chick embryos as an in vivo model. The experiments have shown anti-metastatic activity of POH when the C6 murine glial cells were exposed to a concentration of 0.3 to 0.003% POH for 2 h, prior to its inoculation in chick embryo chorioallantoic membrane model. This phenomenon shows the possible role of POH as an in vivo anti-metastatic drug.

Proinflammatory and cytotoxic effects of hexadecylphosphocholine (miltefosine) against drug-resistant strains of Trypanosoma cruzi.

The increased resistance of the protozoan parasite Trypanosoma cruzi to nitro derivatives is one of the major problems for the successful treatment of Chagas' disease. In the present study, we have tested the effects of 1-O-hexadecylphosphocholine (miltefosine) against strains of T. cruzi that are partially resistant (strain Y) and highly resistant (strain Colombiana) to the drugs in clinical use. As expected, epimastigotes of strain Colombiana showed higher levels of resistance to benznidazole than those of strain Y. However, the level of resistance to miltefosine was the same for both strains. This alkylphospholipid was also extremely toxic against intracellular amastigotes of both strains. This ether-lipid analogue induced in a dose-dependent manner the production of tumor necrosis factor alpha and nitric oxide (NO) radicals by infected and noninfected macrophages, suggesting that miltefosine may activate macrophages in vitro. Nevertheless, the cytotoxic effect of miltefosine against intracellular amastigotes was independent of the amount of NO produced by the infected macrophages since the same dose-response curves for miltefosine were observed when the NO production was blocked by the NO synthase inhibitor N(G)-monomethyl-L-arginine monoacetate. Preliminary in vivo studies with BALB/c mice infected with strain Y indicated that oral miltefosine promoted survival and reduced the parasitemia to levels comparable to those observed when benznidazole was used. Four months after treatment, no parasites were detected in the blood or spleen tissue sections maintained in culture. Together, these results support the hypothesis that miltefosine may be used for the treatment of Chagas' disease, including cases caused by resistant strains of T. cruzi.