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

Alterações comportamentais durante a infecção experimental pelo Trypanosoma cruzi

Since the discovery of Chagas disease in 1909, it is known that the protozoan parasite Trypanosoma cruzi infects the central nervous system (CNS). Neuropathological lesions with parasites in glial cells and, rarely, in CNS neurons are found during the acute phase of infection in humans. Even though neurological alterations are rare in the chronic phase, they seem to be severe in immunocompromised individuals such as those co-infected with human immunodeficiency virus (HIV), transplant or suffering from cancer. Behavioral changes, for instance depression, are detected in patients during chronic T. cruzi infection, but the cause of these disorders is unknown. Experimentally, C3H/He mice develop acute meningoencephalitis that is resolved in the chronic phase, while C57/BL6 mice are resistant to acute meningoencephalitis. However, T. cruzi and its antigens are found in the CNS of mice of both strains in the acute and chronic phases of infection. In this work, we propose to assess whether cognitive (memory/learning), motor and psychological (depression) changes are developed in experimental T. cruzi infection and test the hypothesis that, if present, behavioral alterations are consequences of acute meningoencephalitis. We used C3H/He and C57BL/6 mice strains, respectively, susceptible and resistant to T. cruzi-elicited acute meningoencephalitis. After intraperitoneal infection with 100 blood trypomastigotes of the Colombian T. cruzi strain, the animals were evaluated during the acute (30 dpi) and chronic (90 dpi) phases. The open field test was applied to evaluate locomotor and exploratory activities. Memory was assessed adopting object recognition and passive avoidance tests. The forced swim test and tail suspension were used to assess depression. The results of open field test showed neither exploratory nor motor abnormalities in animals susceptible or resistant to acute meningoencephalitis, during the acute and chronic phases of infection. The results obtained by means of the passive avoidance test suggest that T. cruzi infection does not interfere with aversive conditioning. However, T. cruzi infection of animals of C3H/He and C57BL/6 mice led to significant (p <0.05) alterations in memory recognition pattern, suggesting a mnemonic deficit during T. cruzi infection. Increased immobility time (p <0.0001), assessed by forced swim and tail suspension tests, was observed in infected mice of both C3H/He and C57BL/6 strains, being more expressive in animals of the C3H/He strain. Treatment with the antidepressive fluoxetine revealed improvement in depressive profile detected in infected mice of both strains, reinforcing the existence of behavioral disorders of neurological origin. In conclusion, we suggest that the behavioral changes (memory and learning disabilities as well as depression) are present during experimental T. cruzi infection, independently of the presence of acute meningoencephalitis, therefore not being its consequenceCoordenação de Aperfeiçoamento de Pessoal de Nível SuperiorDesde a descoberta da doença de Chagas em 1909, sabe-se que o Trypanosoma cruzi acomete o sistema nervoso central (SNC). Lesões neuropatológicas como a presença do parasito nas células gliais do SNC de humanos, são encontradas durante a fase aguda da infecção. Na fase crônica, a forma nervosa é rara, sendo grave nos indivíduos imunossuprimidos como: co-infecção pelo HIV, pacientes transplantados ou acometidos de câncer. Alterações comportamentais são detectadas em pacientes durante a fase crônica da infecção, porém a causa desses distúrbios é desconhecida. Experimentalmente, camundongos da linhagem C3H/He desenvolvem intensa meningoencefalite na fase aguda da infecção que se resolve na fase crônica. Embora antígenos parasitários persistam no SNC. Propomos avaliar as alterações cognitivas (memória/aprendizado), motoras e psíquicas (depressão) durante a infecção experimental pelo T. cruzi. Foram utilizados camundongos das linhagens C57BL/6 e C3H/He, resistente e suscetível à meningoencefalite induzida pelo T. cruzi, respectivamente. Após infecção com 100 formas tripomastigotas da cepa Colombiana do T. cruzi, via intraperitoneal, os animais foram avaliados na fase aguda (30 dpi) e na fase cronica (90 dpi). O teste de campo aberto foi aplicado para avaliação da atividade locomotora e exploratória, sendo ainda avaliado a memória através do teste de reconhecimento de objetos e o teste de esquiva passiva. Os testes de nado forçado e suspensão de cauda foram aplicados para avaliar a depressão. Os resultados obtidos no teste campo aberto não mostraram nos animais resistentes ou suscetíveis a meningoencefalite aguda alteração motora e exploratória. Diante dos resultados obtidos com o teste de esquiva passiva sugerimos que a infecção pelo T. cruzi não interfere na memória aversiva. Porém, a infecção em ambas as linhagens de camundongos (C3H/He e C57BL/6) pode alterar a memória de reconhecimento (p<0,05), sugerindo um déficit mnemônico durante a infecção pelo T. cruzi. Os resultados obtidos nos testes de nado forçado e suspensão de cauda mostraram um tempo de imobilidade significativamente maior (p<0,0001), em ambas as linhagens de camundongos, sendo mais expressivo na linhagem C3H/He. O tratamento com anti-depressivo mostrou a melhora do perfil depressivo observado nos animais infectados de ambas as linhagens. Em conclusão sugerimos que as alterações comportamentais (memória/aprendizado e depressão) estão presentes durante a infecção experimental pelo T. cruzi e são independente da presença da meningoencefalite aguda, não sendo, portanto, sequela dest

Severity of chronic experimental Chagas' heart disease parallels tumour necrosis factor and nitric oxide levels in the serum: models of mild and severe disease

Heart tissue inflammation, progressive fibrosis and electrocardiographic alterations occur in approximately 30% of patients infected by Trypanosoma cruzi, 10-30 years after infection. Further, plasma levels of tumour necrosis factor (TNF) and nitric oxide (NO) are associated with the degree of heart dysfunction in chronic chagasic cardiomyopathy (CCC). Thus, our aim was to establish experimental models that mimic a range of parasitological, pathological and cardiac alterations described in patients with chronic Chagas&#8217; heart disease and evaluate whether heart disease severity was associated with increased TNF and NO levels in the serum. Our results show that C3H/He mice chronically infected with the Colombian T. cruzi strain have more severe cardiac parasitism and inflammation than C57BL/6 mice. In addition, connexin 43 disorganisation and fibronectin deposition in the heart tissue, increased levels of creatine kinase cardiac MB isoenzyme activity in the serum and more severe electrical abnormalities were observed in T. cruzi-infected C3H/He mice compared to C57BL/6 mice. Therefore, T. cruzi-infected C3H/He and C57BL/6 mice represent severe and mild models of CCC, respectively. Moreover, the CCC severity paralleled the TNF and NO levels in the serum. Therefore, these models are appropriate for studying the pathophysiology and biomarkers of CCC progression, as well as for testing therapeutic agents for patients with Chagas&#8217; heart disease

Behavioural alterations are independent of sickness behaviour in chronic experimental Chagas disease

Submitted by sandra infurna ([email protected]) on 2016-03-03T16:31:46Z No. of bitstreams: 1 glaucia_pereira_etal_IOC_2015.pdf: 490511 bytes, checksum: 87cc343c1fc67155a453519d8ae9122c (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2016-03-03T16:39:59Z (GMT) No. of bitstreams: 1 glaucia_pereira_etal_IOC_2015.pdf: 490511 bytes, checksum: 87cc343c1fc67155a453519d8ae9122c (MD5)Made available in DSpace on 2016-03-03T16:39:59Z (GMT). No. of bitstreams: 1 glaucia_pereira_etal_IOC_2015.pdf: 490511 bytes, checksum: 87cc343c1fc67155a453519d8ae9122c (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 das Interações. Rio de Janeiro, RJ, Brasil.The existence of the nervous form of Chagas disease is a matter of discussion since Carlos Chagas described neurological disorders, learning and behavioural alterations in Trypanosoma cruzi-infected individuals. In most patients, the clinical manifestations of the acute phase, including neurological abnormalities, resolve spontaneously without apparent consequence in the chronic phase of infection. However, chronic Chagas disease patients have behavioural changes such as psychomotor alterations, attention and memory deficits, and depression. In the present study, we tested whether or not behavioural alterations are reproducible in experimental models. We show that C57BL/6 mice chronically infected with the Colombian strain of T. cruzi (150 days post-infection) exhibit behavioural changes as (i) depression in the tail suspension and forced swim tests, (ii) anxiety analysed by elevated plus maze and open field test sand and (iii) motor coordination in the rotarod test. These alterations are neither associated with neuromuscular disorders assessed by the grip strength test nor with sickness behaviour analysed by temperature variation sand weight loss. Therefore, chronically T. cruzi-infected mice replicate behavioural alterations (depression and anxiety) detected in Chagas disease patients opening an opportunity to study the interconnection and the physiopathology of these two biological processes in an infectious scenario

Behavioural alterations are independent of sickness behaviour in chronic experimental Chagas disease

The existence of the nervous form of Chagas disease is a matter of discussion since Carlos Chagas described neurological disorders, learning and behavioural alterations in Trypanosoma cruzi-infected individuals. In most patients, the clinical manifestations of the acute phase, including neurological abnormalities, resolve spontaneously without apparent consequence in the chronic phase of infection. However, chronic Chagas disease patients have behavioural changes such as psychomotor alterations, attention and memory deficits, and depression. In the present study, we tested whether or not behavioural alterations are reproducible in experimental models. We show that C57BL/6 mice chronically infected with the Colombian strain of T. cruzi (150 days post-infection) exhibit behavioural changes as (i) depression in the tail suspension and forced swim tests, (ii) anxiety analysed by elevated plus maze and open field test sand and (iii) motor coordination in the rotarod test. These alterations are neither associated with neuromuscular disorders assessed by the grip strength test nor with sickness behaviour analysed by temperature variation sand weight loss. Therefore, chronically T. cruzi-infected mice replicate behavioural alterations (depression and anxiety) detected in Chagas disease patients opening an opportunity to study the interconnection and the physiopathology of these two biological processes in an infectious scenario

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

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