A human type 5 adenovirus-based Trypanosoma cruzi therapeutic vaccine re-programs immune response and reverses chronic cardiomyopathy

Chagas disease (CD), caused by the protozoan Trypanosoma cruzi, is a prototypical neglected tropical disease. Specific immunity promotes acute phase survival. Nevertheless, one-third of CD patients develop chronic chagasic cardiomyopathy (CCC) associated with parasite persistence and immunological unbalance. Currently, the therapeutic management of patients only mitigates CCC symptoms. Therefore, a vaccine arises as an alternative to stimulate protective immunity and thereby prevent, delay progression and even reverse CCC. We examined this hypothesis by vaccinating mice with replication-defective human Type 5 recombinant adenoviruses (rAd) carrying sequences of amastigote surface protein-2 (rAdASP2) and trans-sialidase (rAdTS) T. cruzi antigens. For prophylactic vaccination, naive C57BL/6 mice were immunized with rAdASP2+rAdTS (rAdVax) using a homologous prime/boost protocol before challenge with the Colombian strain. For therapeutic vaccination, rAdVax administration was initiated at 120 days post-infection (dpi), when mice were afflicted by CCC. Mice were analyzed for electrical abnormalities, immune response and cardiac parasitism and tissue damage. Prophylactic immunization with rAdVax induced antibodies and H-2Kb-restricted cytotoxic and interferon (IFN)gamma-producing CD8+ T-cells, reduced acute heart parasitism and electrical abnormalities in the chronic phase. Therapeutic vaccination increased survival and reduced electrical abnormalities after the prime (analysis at 160 dpi) and the boost (analysis at 180 and 230 dpi). Post-therapy mice exhibited less heart injury and electrical abnormalities compared with pre-therapy mice. rAdVax therapeutic vaccination preserved specific IFNgamma-mediated immunity but reduced the response to polyclonal stimuli (anti-CD3 plus anti-CD28), CD107a+ CD8+ T-cell frequency and plasma nitric oxide (NO) levels. Moreover, therapeutic rAdVax reshaped immunity in the heart tissue as reduced the number of perforin+ cells, preserved the number of IFNgamma+ cells, increased the expression of IFNgamma mRNA but reduced inducible NO synthase mRNA. Vaccine-based immunostimulation with rAd might offer a rational alternative for re-programming the immune response to preserve and, moreover, recover tissue injury in Chagas\u27 heart disease

Towards the establishment of a single standard curve for quantification of Trypanosoma cruzi natural populations using a synthetic satellite unit DNA sequence

Accurate diagnostics tools and surrogate markers of parasitological response to treatment are priority needs for management of Chagas disease. Quantitative real-time PCR (qPCR) is used for treatment monitoring, but variability in copy dosage and sequences of molecular target genes among different Trypanosoma cruzi strains limit the precision of quantitative measures. To improve qPCR quantification accuracy, we designed and evaluated a synthetic DNA molecule containing a Satellite DNA (satDNA) repeat unit as standard for quantification of T. cruzi loads in clinical samples, independently of the parasite strain. Probit regression analysis established for Dm28c (Tc I) and CL-Brener (Tc VI) stocks similar LOD95 values (0.903 (0.745-1.497) and 0.667 (CI 0.113-3.927) copy numbers/μL, respectively), when synthetic DNA was the standard for quantification, thus allowing direct comparison of loads in samples infected with different DTUs. This standard curve was evaluated in 205 samples from 38 acute oral and 19 chronic CD patients from different geographical areas infected with different genotypes, including samples obtained during treatment follow-up, and high agreement with parasitic load trends using standard curves based on DNA extracted from spiked blood with counted parasites was obtained. This qPCR-based quantification strategy will be a valuable tool in phase III clinical trials, to follow-up patients under treatment or at risk of reactivation and in experimental models using different parasite strains.Fil: Muñoz Calderon, Arturo Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Silva Gomess, Natalia Lins. Fundación Oswaldo Cruz; BrasilFil: Apodaca, Sofia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Alarcón de Noya, Belkisyolé. Universidad Central de Venezuela; VenezuelaFil: Díaz Bello, Zoraida. Universidad Central de Venezuela; VenezuelaFil: Quintino Souza, Leticia Rocha. Fundación Oswaldo Cruz; BrasilFil: Tavares Costa, Alexandre Dias. Fundación Oswaldo Cruz; BrasilFil: Britto, Constança. Fundación Oswaldo Cruz; BrasilFil: Moreira, Otacilio Cruz. Fundación Oswaldo Cruz; BrasilFil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

Validación analítica de métodos cuantitativos de PCR en tiempo real para la cuantificación del ADN de Trypanosoma cruzi en muestras de sangre de pacientes con enfermedad de Chagas

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.

Validación analítica de métodos cuantitativos de PCR en tiempo real para la cuantificación del ADN de Trypanosoma cruzi en muestras de sangre de pacientes con enfermedad de Chagas

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.

Development of real-time PCR assays for evaluation of immune response and parasite load in golden hamster (Mesocricetus auratus) infected by Leishmania (Viannia) braziliensis

Submitted by Sandra Infurna ([email protected]) on 2017-02-23T15:38:26Z No. of bitstreams: 1 octacilio_moreira_etal_IOC_2016.pdf: 3840446 bytes, checksum: 0d27fcfdafc79080420cd9236cca192e (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2017-02-23T15:48:47Z (GMT) No. of bitstreams: 1 octacilio_moreira_etal_IOC_2016.pdf: 3840446 bytes, checksum: 0d27fcfdafc79080420cd9236cca192e (MD5)Made available in DSpace on 2017-02-23T15:48:47Z (GMT). No. of bitstreams: 1 octacilio_moreira_etal_IOC_2016.pdf: 3840446 bytes, checksum: 0d27fcfdafc79080420cd9236cca192e (MD5) Previous issue date: 2016Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório Interdisciplinar de Pesquisas Médicas. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório Interdisciplinar de Pesquisas Médicas. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório Interdisciplinar de Pesquisas Médicas. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório Interdisciplinar de Pesquisas Médicas. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ. Brasil.Cutaneous leishmaniasis (CL) is a neglected disease with a broad spectrum of clinical manifestations, ranging from small cutaneous nodules to severe mucosal tissue destruction. Leishmania (Viannia) braziliensis is the main species attributed to CL in the Americas. However, studies of experimental infection are limited in the murine model due to the self-resolutive pattern of the disease. Previously, our group demonstrated that the hamster model reproduces many of the clinical and histopathological features observed in humans. Herein, we standardized a RT-qPCR gene expression assay to evaluate a panel of immunological markers and a qPCR assay in order to quantify with high sensitivity and reproducibility the parasite load in skin lesions

Pertya ovata Maxim.

原著和名: カウヤバウキ科名: キク科 = Compositae採集地: 福島県 西白河郡 小田川村 (岩代 西白河郡 小田川村)採集日: 1945/10/13採集者: 萩庭丈壽整理番号: JH047074国立科学博物館整理番号: TNS-VS-99707

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

Submitted by Sandra Infurna ([email protected]) on 2016-12-06T14:31:36Z No. of bitstreams: 1 glaucia_pereira_etal_IOC_2016.pdf: 1715795 bytes, checksum: 37fc81d5397e81ab69a5272837cf7d49 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2016-12-06T14:46:08Z (GMT) No. of bitstreams: 1 glaucia_pereira_etal_IOC_2016.pdf: 1715795 bytes, checksum: 37fc81d5397e81ab69a5272837cf7d49 (MD5)Made available in DSpace on 2016-12-06T14:46:08Z (GMT). No. of bitstreams: 1 glaucia_pereira_etal_IOC_2016.pdf: 1715795 bytes, checksum: 37fc81d5397e81ab69a5272837cf7d49 (MD5) Previous issue date: 2016Fundação 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 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 Fluminense. Faculdade de Medicina. 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 das Interações. Rio de Janeiro, RJ, Brasil.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

Sphingosine-1-Phosphate Receptor 1 Is Involved in Non-Obese Diabetic Mouse Thymocyte Migration Disorders

NOD (non-obese diabetic) mice spontaneously develop type 1 diabetes following T cell-dependent destruction of pancreatic β cells. Several alterations are observed in the NOD thymus, including the presence of giant perivascular spaces (PVS) filled with single-positive (SP) CD4+ and CD8+ T cells that accumulate in the organ. These cells have a decreased expression of membrane CD49e (the α5 integrin chain of the fibronectin receptor VLA-5 (very late antigen-5). Herein, we observed lower sphingosine-1-phosphate receptor 1 (S1P1) expression in NOD mouse thymocytes when compared with controls, mainly in the mature SP CD4+CD62Lhi and CD8+CD62Lhi subpopulations bearing the CD49e− phenotype. In contrast, differences in S1P1 expression were not observed in mature CD49e+ thymocytes. Functionally, NOD CD49e− thymocytes had reduced S1P-driven migratory response, whereas CD49e+ cells were more responsive to S1P. We further noticed a decreased expression of the sphingosine-1-phosphate lyase (SGPL1) in NOD SP thymocytes, which can lead to a higher sphingosine-1-phosphate (S1P) expression around PVS and S1P1 internalization. In summary, our results indicate that the modulation of S1P1 expression and S1P/S1P1 interactions in NOD mouse thymocytes are part of the T-cell migratory disorder observed during the pathogenesis of type 1 diabetes

Sphingosine-1-Phosphate Induces Dose-Dependent Chemotaxis or Fugetaxis of T-ALL Blasts through S1P1 Activation

Submitted by Sandra Infurna ([email protected]) on 2016-12-29T14:03:06Z No. of bitstreams: 1 carolina_messias_etal_IOC_2016.pdf: 2089682 bytes, checksum: 1a0d7c222dd6d80e2d67efd664f2b0a0 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2016-12-29T14:17:22Z (GMT) No. of bitstreams: 1 carolina_messias_etal_IOC_2016.pdf: 2089682 bytes, checksum: 1a0d7c222dd6d80e2d67efd664f2b0a0 (MD5)Made available in DSpace on 2016-12-29T14:17:22Z (GMT). No. of bitstreams: 1 carolina_messias_etal_IOC_2016.pdf: 2089682 bytes, checksum: 1a0d7c222dd6d80e2d67efd664f2b0a0 (MD5) Previous issue date: 2016Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa sobre o Timo. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa sobre o Timo. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa sobre o Timo. 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 sobre o Timo. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa sobre o Timo. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa sobre o Timo. Rio de Janeiro, RJ, Brasil.Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid involved in several physiological processes including cell migration and differentiation. S1P signaling is mediated through five G protein-coupled receptors (S1P1-S1P5). S1P1 is crucial to the exit of T-lymphocytes from the thymus and peripheral lymphoid organs through a gradient of S1P. We have previously observed that T-ALL and T-LBL blasts express S1P1. Herein we analyzed the role of S1P receptors in the migratory pattern of human T-cell neoplastic blasts. S1P-triggered cell migration was directly related to S1P1 expression. T-ALL blasts expressing low levels of S1P1 mRNA (HPB-ALL) did not migrate toward S1P, whereas those expressing higher levels of S1P1 (MOLT-4, JURKAT and CEM) did migrate. The S1P ligand induced T-ALL cells chemotaxis in concentrations up to 500 nM and induced fugetaxis in higher concentrations (1000-10000 nM) through interactions with S1P1. When S1P1 was specifically blocked by the W146 compound, S1P-induced migration at lower concentrations was reduced, whereas higher concentrations induced cell migration. Furthermore, we observed that S1P/S1P1 interactions induced ERK and AKT phosphorylation, and modulation of Rac1 activity. Responding T-ALL blasts also expressed S1P3 mRNA but blockage of this receptor did not modify migratory responses. Our results indicate that S1P is involved in the migration of T-ALL/LBL blasts, which is dependent on S1P1 expression. Moreover, S1P concentrations in the given microenvironment might induce dose-dependent chemotaxis or fugetaxis of T-ALL blasts