Cytokine Production but Lack of Proliferation in Peripheral Blood Mononuclear Cells from Chronic Chagas' Disease Cardiomyopathy Patients in Response to T. cruzi Ribosomal P Proteins

Background:Trypanosoma cruzi ribosomal P proteins, P2β and P0, induce high levels of antibodies in patients with chronic Chagas' disease Cardiomyopathy (CCC). It is well known that these antibodies alter the beating rate of cardiomyocytes and provoke apoptosis by their interaction with β1-adrenergic and M2-muscarinic cardiac receptors. Based on these findings, we decided to study the cellular immune response to these proteins in CCC patients compared to non-infected individuals.Methodology/Principal findings:We evaluated proliferation, presence of surface activation markers and cytokine production in peripheral blood mononuclear cells (PBMC) stimulated with P2β, the C-terminal portion of P0 (CP0) proteins and T. cruzi lysate from CCC patients predominantly infected with TcVI lineage. PBMC from CCC patients cultured with P2β or CP0 proteins, failed to proliferate and express CD25 and HLA-DR on T cell populations. However, multiplex cytokine assays showed that these antigens triggered higher secretion of IL-10, TNF-α and GM-CSF by PBMC as well as both CD4+ and CD8+ T cells subsets of CCC subjects. Upon T. cruzi lysate stimulation, PBMC from CCC patients not only proliferated but also became activated within the context of Th1 response. Interestingly, T. cruzi lysate was also able to induce the secretion of GM-CSF by CD4+ or CD8+ T cells.Conclusions/Significance:Our results showed that although the lack of PBMC proliferation in CCC patients in response to ribosomal P proteins, the detection of IL-10, TNF-α and GM-CSF suggests that specific T cells could have both immunoregulatory and pro-inflammatory potential, which might modulate the immune response in Chagas' disease. Furthermore, it was possible to demonstrate for the first time that GM-CSF was produced by PBMC of CCC patients in response not only to recombinant ribosomal P proteins but also to parasite lysate, suggesting the value of this cytokine to evaluate T cells responses in T. cruzi infection.Fil: Longhi, Silvia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Atienza, Augusto. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; ArgentinaFil: Perez Prados, Graciela. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Juan A. Fernández"; ArgentinaFil: Buying, Alcinette. Torrey Pines Institute for Molecular Studies; Estados UnidosFil: Balouz, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Buscaglia, Carlos Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Santos, Radleigh. Torrey Pines Institute for Molecular Studies; Estados UnidosFil: Tasso, Laura Mónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Bonato, Ricardo. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; ArgentinaFil: Chiale, Pablo. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; ArgentinaFil: Pinilla, Clemencia. Torrey Pines Institute for Molecular Studies; Estados UnidosFil: Judkowski, Valeria A.. Torrey Pines Institute for Molecular Studies; Estados UnidosFil: Gomez, Karina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentin

Chagas' disease: an update on immune mechanisms and therapeutic strategies

The final decade of the 20th century was marked by an alarming resurgence in infectious diseases caused by tropical parasites belonging to the kinetoplastid protozoan order. Among the pathogenic trypanosomatids, some species are of particular interest due to their medical importance. These species include the agent responsible for Chagas' disease, Trypanosoma cruzi. Approximately 8 to 10 million people are infected in the Americas, and approximately 40 million are at risk. in the present review, we discuss in detail the immune mechanisms elicited during infection by T. cruzi and the effects of chemotherapy in controlling parasite proliferation and on the host immune system.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Instituto Nacional de Biologia Estrutural e QuImica Medicinal em Doencas Infecciosas (INBEQMeDI)Univ São Paulo, Inst Biomed Sci, Dept Parasitol, São Paulo, BrazilMackenzie Presbeterian Univ, Ctr Biomol Sci & Hlth, São Paulo, BrazilNatl Univ Rosario, Sch Med Sci, Inst Immunol, Rosario, Santa Fe, ArgentinaCSIC, Inst Parasitol & Biomed Lopez Neyra, Granada, SpainUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilFAPESP: 08/57596-4FAPESP: 07/08648-9CNPq: 473906/2008-2Web of Scienc

Immunological imbalance between IFN-³ and IL-10 levels in the sera of patients with the cardiac form of Chagas disease

The immune response is crucial for protection against disease; however, immunological imbalances can lead to heart and digestive tract lesions in chagasic patients. Several studies have evaluated the cellular and humoral immune responses in chagasic patients in an attempt to correlate immunological findings with clinical forms of Chagas disease. Moreover, immunoglobulins and cytokines are important for parasitic control and are involved in lesion genesis. Here, cytokine and IgG isotype production were studied, using total epimastigote antigen on sera of chagasic patients with indeterminate (IND, n = 27) and cardiac (CARD, n = 16) forms of the disease. Samples from normal, uninfected individuals (NI, n = 30) were use as controls. The results showed that sera from both IND and CARD patients contained higher levels of Trypanosoma cruzi-specific IgG1 (IgG1) antibodies than sera from NI. No difference in IgG2 production levels was observed between NI, IND and CARD patients, nor was a difference in IL-10 and IFN-³ production detected in the sera of IND, CARD and NI patients. However, IND patients displayed a positive correlation between IL-10 and IFN-³ levels in serum, while CARD patients showed no such correlation, indicating an uncontrolled inflammatory response in CARD patients. These findings support the hypothesis that a lack of efficient regulation between IFN-³ and IL-10 productions in CARD patients may lead to cardiac immunopathology.CNP

Perfil clínico e epidemiológico de pacientes idosos com doença de Chagas atendidos entre 2005-2013 por um serviço de atenção farmacêutica no estado do Ceará, nordeste do Brasil

Controlando-se a transmissão da doença de Chagas, surge o desafio de prestar assistência a milhões de pacientes infectados que chegam à velhice. Neste estudo, foram avaliados os registros socioeconômicos, demográficos e de comorbidades de todos os pacientes chagásicos idosos acompanhados no Serviço de Atenção Farmacêutica do Laboratório de Pesquisa em Doença de Chagas. As informações relacionadas à forma clínica da doença foram obtidas a partir de registros médicos disponibilizados pelo Hospital Universitário Walter Cantídio. O perfil da população estudada foi de: mulheres (50,5%); idade média de 67 anos; aposentados (54,6%); casados (51,6%); alta taxa de analfabetismo (40,2%); e renda familiar de um salário mínimo (51,5%). As formas clínicas predominantes da doença de Chagas foram a cardíaca (65,3%) e a indeterminada (14,7%). As principais alterações eletrocardiográficas foram o bloqueio de ramo direito (41,0%), associado ou não ao bloqueio ântero superior esquerdo (27,4%). O número médio de comorbidades por paciente foi de 2,23 ± 1,54, sendo a hipertensão arterial sistêmica a principal encontrada (67,0%). Verificou-se que os idosos constituem grupo vulnerável de pacientes que associam o envelhecimento com as alterações cardíacas e/ou digestivas resultantes da evolução da doença de Chagas e outras comorbidades, o que exige atenção especial dos serviços de saúde para um atendimento médico e social mais adequado.By controlling the transmission of Chagas disease, the challenge of providing assistance to millions of infected patients that reach old age arises. In this study, the socioeconomic, demographic and comorbidity records of all elderly chagasic patients followed at the Pharmaceutical Care Service of the Chagas Disease Research Laboratory were assessed. The information related to the clinical form of the disease was obtained from medical records provided by the Walter Cantídio University Hospital. The profile of the studied population was: women (50.5%); mean age of 67 years; retired (54.6%); married (51.6 %); high illiteracy rate (40.2%); and family income equal to the minimum wage (51.5%). The predominant clinical forms of Chagas disease were cardiac (65.3%) and indeterminate (14.7%). The main electrocardiographic changes were the right bundle branch block (41.0%), associated or not with the anterosuperior left bundle branch block (27.4%). The average number of comorbidities per patient was 2.23 ± 1.54, with systemic arterial hypertension being the main one found (67.0%). It was found that the elderly comprise a vulnerable group of patients that associate aging with cardiac and/or digestive disorders resulting from the evolution of Chagas disease and other comorbidities, which requires special attention from health services to ensure more appropriate medical and social care

CD8+ T-Cells Expressing Interferon Gamma or Perforin Play Antagonistic Roles in Heart Injury in Experimental Trypanosoma Cruzi-Elicited Cardiomyopathy

In Chagas disease, CD8+ T-cells are critical for the control of Trypanosoma cruzi during acute infection. Conversely, CD8+ T-cell accumulation in the myocardium during chronic infection may cause tissue injury leading to chronic chagasic cardiomyopathy (CCC). Here we explored the role of CD8+ T-cells in T. cruzi-elicited heart injury in C57BL/6 mice infected with the Colombian strain. Cardiomyocyte lesion evaluated by creatine kinase-MB isoenzyme activity levels in the serum and electrical abnormalities revealed by electrocardiogram were not associated with the intensity of heart parasitism and myocarditis in the chronic infection. Further, there was no association between heart injury and systemic anti-T. cruzi CD8+ T-cell capacity to produce interferon-gamma (IFNγ) and to perform specific cytotoxicity. Heart injury, however, paralleled accumulation of anti-T. cruzi cells in the cardiac tissue. In T. cruzi infection, most of the CD8+ T-cells segregated into IFNγ+ perforin (Pfn)neg or IFNγnegPfn+ cell populations. Colonization of the cardiac tissue by anti-T. cruzi CD8+Pfn+ cells paralleled the worsening of CCC. The adoptive cell transfer to T. cruzi-infected cd8−/− recipients showed that the CD8+ cells from infected ifnγ−/−pfn+/+ donors migrate towards the cardiac tissue to a greater extent and caused a more severe cardiomyocyte lesion than CD8+ cells from ifnγ+/+pfn−/− donors. Moreover, the reconstitution of naïve cd8−/− mice with CD8+ cells from naïve ifnγ+/+pfn−/− donors ameliorated T. cruzi-elicited heart injury paralleled IFNγ+ cells accumulation, whereas reconstitution with CD8+ cells from naïve ifnγ−/−pfn+/+ donors led to an aggravation of the cardiomyocyte lesion, which was associated with the accumulation of Pfn+ cells in the cardiac tissue. Our data support a possible antagonist effect of CD8+Pfn+ and CD8+IFNγ+ cells during CCC. CD8+IFNγ+ cells may exert a beneficial role, whereas CD8+Pfn+ may play a detrimental role in T. cruzi-elicited heart injury

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

The Thymus Is a Common Target Organ in Infectious Diseases

Infectious disease immunology has largely focused on the effector immune response, changes in the blood and peripheral lymphoid organs of infected individuals, and vaccine development. Studies of the thymus in infected individuals have been neglected, although this is progressively changing. The thymus is a primary lymphoid organ, able to generate mature T cells that eventually colonize secondary lymphoid organs, and is therefore essential for peripheral T cell renewal. Recent data show that normal thymocyte development and export can be altered as a result of an infectious disease. One common feature is the severe atrophy of the infected organ, mainly due to the apoptosis-related depletion of immature CD4(+)CD8(+) thymocytes. Additionally, thymocyte proliferation is frequently diminished. The microenvironmental compartment of the thymus is also affected, particularly in acute infectious diseases, with a densification of the epithelial network and an increase in the deposition of extracellular matrix. In the murine model of Chagas disease, intrathymic chemokine production is also enhanced, and thymocytes from Trypanosoma cruzi-infected mice exhibit greater numbers of cell migration-related receptors for chemokines and extracellular matrix, as well as increased migratory responses to the corresponding ligands. This profile is correlated with the appearance of potentially autoreactive thymus-derived immature CD4(+)CD8(+) T cells in peripheral organs of infected animals. A variety of infectious agents—including viruses, protozoa, and fungi—invade the thymus, raising the hypothesis of the generation of central immunological tolerance for at least some of the infectious agent-derived antigens. It seems clear that the thymus is targeted in a variety of infections, and that such targeting may have consequences on the behavior of peripheral T lymphocytes. In this context, thymus-centered immunotherapeutic approaches potentially represent a new tool for the treatment of severe infectious diseases

NADPH Phagocyte Oxidase Knockout Mice Control Trypanosoma cruzi Proliferation, but Develop Circulatory Collapse and Succumb to Infection

•NO is considered to be a key macrophage-derived cytotoxic effector during Trypanosoma cruzi infection. On the other hand, the microbicidal properties of reactive oxygen species (ROS) are well recognized, but little importance has been attributed to them during in vivo infection with T. cruzi. In order to investigate the role of ROS in T. cruzi infection, mice deficient in NADPH phagocyte oxidase (gp91phox−/− or phox KO) were infected with Y strain of T. cruzi and the course of infection was followed. phox KO mice had similar parasitemia, similar tissue parasitism and similar levels of IFN-γ and TNF in serum and spleen cell culture supernatants, when compared to wild-type controls. However, all phox KO mice succumbed to infection between day 15 and 21 after inoculation with the parasite, while 60% of wild-type mice were alive 50 days after infection. Further investigation demonstrated increased serum levels of nitrite and nitrate (NOx) at day 15 of infection in phox KO animals, associated with a drop in blood pressure. Treatment with a NOS2 inhibitor corrected the blood pressure, implicating NOS2 in this phenomenon. We postulate that superoxide reacts with •NO in vivo, preventing blood pressure drops in wild type mice. Hence, whilst superoxide from phagocytes did not play a critical role in parasite control in the phox KO animals, its production would have an important protective effect against blood pressure decline during infection with T. cruzi