Inducible Nitric Oxide Synthase in Heart Tissue and Nitric Oxide in Serum of Trypanosoma cruzi-Infected Rhesus Monkeys: Association with Heart Injury

Chagas disease, a neglected tropical disease caused by the protozoan Trypanosoma cruzi, afflicts from 8 to 15 million people in the Latin America. Chronic chagasic cardiomyopathy (CCC) is the most frequent manifestation of Chagas disease. Currently, patient management only mitigates CCC symptoms. The pathogenic factors leading to CCC remain unknown; therefore their comprehension may contribute to develop more efficient therapies. In patients, high nitric oxide (NO) levels have been associated with CCC severity. In T. cruzi-infected mice, NO, mainly produced via inducible nitric oxide synthase (iNOS/NOS2), is proposed to work in parasite control. However, the participation of iNOS/NOS2 and NO in T. cruzi control and heart injury has been questioned. Here, infected rhesus monkeys and iNOS/NOS2-deficient mice were used to explore the participation of iNOS/NOS2-derived NO in heart injury in T. cruzi infection. Chronically infected monkeys presented electrical abnormalities, myocarditis and fibrosis, resembling the spectrum of human CCC. Moreover, cardiomyocyte lesion correlated with iNOS/NOS2+ cells infiltrating the cardiac tissue. Our findings support that parasite-driven iNOS/NOS2+ cells accumulation in the cardiac tissue and NO overproduction contribute to cardiomyopathy severity, mainly disturbing the pathway involved in electrical synchrony in T. cruzi infection

Tumor Necrosis Factor Is a Therapeutic Target for Immunological Unbalance and Cardiac Abnormalities in Chronic Experimental Chagas’ Heart Disease

Made available in DSpace on 2015-05-04T16:34:35Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) constança_britto2etal_IOC_2014.pdf: 2413422 bytes, checksum: 11a863f3310e32c54d8b8d9a64c894e8 (MD5) Previous issue date: 2014Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia das Interações. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia das Interações. Rio de Janeiro, RJ, Brasil.Universidade Federal Fluminense. Departamento de Patologia. Niterói, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia das Interações. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia das Interações. Rio de Janeiro, RJ, Brasil.Background. Chagas disease (CD) is characterized by parasite persistence and immunological unbalance favoring systemic inflammatory profile. Chronic chagasic cardiomyopathy, the main manifestation of CD, occurs in a TNF-enriched milieu and frequently progresses to heart failure. Aimof the Study. To challenge the hypothesis that TNF plays a key role in Trypanosoma cruziinduced immune deregulation and cardiac abnormalities, we tested the effect of the anti-TNF antibody Infliximab in chronically T. cruzi-infected C57BL/6 mice, a model with immunological, electrical, and histopathological abnormalities resembling Chagas’ heart disease. Results. Infliximab therapy did not reactivate parasite but reshaped the immune response as reduced TNF mRNA expression in the cardiac tissue and plasma TNF and IFN levels; diminished the frequency of IL-17A+ but increased IL-10+ CD4+ T-cells; reduced TNF+ but augmented IL-10+ Ly6C+ and F4/80+ cells. Further, anti-TNF therapy decreased cytotoxic activity but preserved IFN-producing VNHRFTLV-specific CD8+ T-cells in spleen and reduced the number of perforin+ cells infiltrating the myocardium. Importantly, Infliximab reduced the frequency of mice afflicted by arrhythmias and second degree atrioventricular blocks and decreased fibronectin deposition in the cardiac tissue. Conclusions. Our data support that TNF is a crucial player in the pathogenesis of Chagas’ heart disease fueling immunological unbalance which contributes to cardiac abnormalities

Pentoxifylline Reverses Chronic Experimental Chagasic Cardiomyopathy in Association with Repositioning of Abnormal CD8+ T-Cell Response

Submitted by sandra infurna ([email protected]) on 2016-04-28T14:57:30Z No. of bitstreams: 1 isabela_pereira_etal_IOC_2015.pdf: 6043084 bytes, checksum: 03af83923a25cfd4e95c3be608b6e3b2 (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2016-04-28T15:07:54Z (GMT) No. of bitstreams: 1 isabela_pereira_etal_IOC_2015.pdf: 6043084 bytes, checksum: 03af83923a25cfd4e95c3be608b6e3b2 (MD5)Made available in DSpace on 2016-04-28T15:07:54Z (GMT). No. of bitstreams: 1 isabela_pereira_etal_IOC_2015.pdf: 6043084 bytes, checksum: 03af83923a25cfd4e95c3be608b6e3b2 (MD5) Previous issue date: 2015Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia das Interações. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia das Interações. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Cardiologia Celular e Molecular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia das Interações. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia das Interações. Rio de Janeiro, RJ, Brasil.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

Nucleoside triphosphate diphosphohydrolase1 (TcNTPDase-1) gene expression is increased due to heat shock and in infective forms of Trypanosoma cruzi

Made available in DSpace on 2015-04-17T17:04:34Z (GMT). No. of bitstreams: 3 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) natalia_silvagomesetal_IOC_2014.pdf: 520023 bytes, checksum: 30bf03d816ed2422bc7422552d6d795b (MD5) 1_natalia_silvagomesetal_IOC_2014.pdf: 520023 bytes, checksum: 30bf03d816ed2422bc7422552d6d795b (MD5) Previous issue date: 2014Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratorio de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratorio de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratorio de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Celular. Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Celular. Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Celular. Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratorio de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ, Brasil.Background: Ecto-Nucleoside Triphosphate Diphosphohydrolases (Ecto-NTPDases) are enzymes that hydrolyze tri- and/or di-phosphate nucleotides. Evidences point to their participation in Trypanosoma cruzi virulence and infectivity. In this work, we evaluate TcNTPDase-1 gene expression in comparison with ecto-NTPDase activity, in order to study the role of TcNTPDase-1 in parasite virulence, infectivity and adaptation to heat shock. Findings: Comparison between distinct T. cruzi isolates (Y, 3663 and 4167 strains, and Dm28c, LL014 and CL-14 clones) showed that TcNTPDase-1 expression was 7.2 ± 1.5 times higher in the Dm28c than the CL-14 avirulent clone. A remarkable expression increase was also observed in the trypomastigote and amastigote forms (22.5 ± 5.6 and 16.3 ± 3.8 times higher than epimastigotes, respectively), indicating that TcNTPDase-1 is overexpressed in T. cruzi infective forms. Moreover, heat shock and long-term cultivation also induced a significant increment on TcNTPDase-1 expression. Conclusions: Our results suggest that TcNTPDase-1 plays an important role on T. cruzi infectivity and adaptation to stress conditions, such as long-term cultivation and heat shock

Analytical validation of real-time quantitative PCR assays for optimum diagnosis of vivax malaria

Submitted by Sandra Infurna ([email protected]) on 2019-08-22T17:36:24Z No. of bitstreams: 1 AlineLavigne_OtacilioMoreira_etal_IOC_2019.pdf: 458878 bytes, checksum: 4993b198c5212641c504f2cc9791d069 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2019-08-22T17:42:31Z (GMT) No. of bitstreams: 1 AlineLavigne_OtacilioMoreira_etal_IOC_2019.pdf: 458878 bytes, checksum: 4993b198c5212641c504f2cc9791d069 (MD5)Made available in DSpace on 2019-08-22T17:42:31Z (GMT). No. of bitstreams: 1 AlineLavigne_OtacilioMoreira_etal_IOC_2019.pdf: 458878 bytes, checksum: 4993b198c5212641c504f2cc9791d069 (MD5) Previous issue date: 2019Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa em Malária. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa em Malária. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Genômica Funcional e Bioinformática. Rio de Janeiro, RJ, Brasil.Universidade do Estado do Rio de Janeiro. Instituto de Medicina Social. Departamento de Epidemiologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa em Malária. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa em Malária. Rio de Janeiro, RJ, Brasil.BACKGROUND The prompt diagnosis of plasmodial species for effective treatment prevents worsening of individual health and avoids transmission maintenance or even malaria reintroduction in areas where Plasmodium does not exist. Polymerase chain reaction (PCR) allows for the detection of parasites below the threshold of microscopic examination. OBJECTIVE Our aim was to develop a real-time PCR test to reduce diagnostic errors and increase efficacy. METHODS The lower limit of quantification and the linearity/analytical sensitivity to measure sensitivity or limit of detection (LoD) were determined. Intra-assay variations (repeatability) and alterations between assays, operators, and instruments (reproducibility) were also assessed to set precision. FINDINGS The linearity in SYBR™ Green and TaqMan™ systems was 106 and 102 copies and analytical sensitivity 1.13 and 1.17 copies/μL, respectively. Real-time PCR was more sensitive than conventional PCR, showing a LoD of 0.01 parasite (p)/μL. Reproducibility and repeatability (precision) were 100% for up to 0.1 p/μL in SYBR™ Green and 1 p/μL in TaqMan™ and conventional PCR. CONCLUSION Real-time PCR may replace conventional PCR in reference laboratories for P. vivax detection due to its rapidity. The TaqMan™ system is the most indicated when quantification assays are required. Performing tests in triplicate when diagnosing Plasmodium-infected-asymptomatic individuals is recommended to minimise diagnostic errors

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

Cytokine mRNA expression in skin of hamsters immunised with <i>L. amazonensis</i> antigens (LaAg) and infected with <i>L. braziliensis</i>.

<p>Golden hamsters (6–7 per group) were immunised with IN LaAg (•), IM LaAg (▴) or PBS (□). Two weeks after the last immunisation, hamsters were challenged with <i>L. braziliensis</i>. (A) mRNA gene expression for IFN-γ and IL-10 in skin lesions was determined 114 days post-infection. (B) Correlation between lesion thickness and IFN-γ (r = 0.76, p<0.0001) or IL-10 (r = 0.68, p<0.0001). Each point represents one animal. The horizontal bars represent the median values. Symbols represent the fold change in cytokine expression relative to that of uninfected hamsters. Data shown are from one of two independent experiments.</p