Caracterización molecular de mecanismos inflamatorios que contribuyen al desarrollo de miocardiopatía en la enfermedad de Chagas

Chagas disease, caused by the parasite Trypanosoma cruzi, is characterized by an intense inflammatory cardiomyopathy, whose pathophysiology is still not fully understood.\nOur study goal was to examine the contribution of diverse mediators of inflammation to the immunopathogenesis of Chagasic cardiomyopathy and also to evaluate the modulatory effects of etiological treatment with benznidazole on inflammatory agents and heart pathology. We investigated novel potential cardiopathogenic factors that remained unexplored so far (as the proinflammatory cytokine MIF), analyzing its biological significance in experimentally infected animals as well as in Chagas disease patients with different myocardial compromise severity grade.\nThe overall results show that proinflammatory cytokines and chemokines together with their specific receptors participate in immunopathogenic mechanisms leading to chronic Chagasic cardiomyopathy. We focused particularly on the dual role of MIF: displaying key immunoprotective features during the initial steps of T. cruzi infection and, through its persistent induction, also being associated with progression towards the most serious forms of heart dysfunction elicited by prolonged Chagas disease. The immunomodulatory ability exhibited by benznidazol therapy administered during the chronic phase of parasite infection could be helpful in balancing MIF levels and reducing inflammatory injury at this stage, thus limiting the risk of long-term cardiovascular damage.Fil: Cutrullis, Romina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaLa enfermedad de Chagas, causada por el parásito Trypanosoma cruzi, se caracteriza por una intensa miocardiopatía inflamatoria, cuya fisiopatogenia aún no se conoce acabadamente.\nNuestro objetivo fue examinar la contribución de distintos mediadores inflamatorios a la inmunopatogénesis de la miocardiopatía chagásica y evaluar el efecto modulador que el tratamiento etiológico con benznidazol pudiera ejercer sobre estos mediadores y la patología inflamatoria. Estudiamos factores potenciales de patogenia chagásica inexplorados, como la citoquina proinflamatoria MIF, analizando su relevancia biológica en el modelo animal y en pacientes infectados con distinto grado de compromiso cardíaco.\nLos resultados obtenidos indican que las citoquinas y quimioquinas proinflamatorias y sus receptores específicos participan en los mecanismos inmunopatogénicos de la miocardiopatía chagásica crónica. Particularmente, destacamos el rol de MIF que, más allá de resultar fundamental para la protección en la etapa aguda de la infección por T. cruzi, mediante su persistente inducción se asocia con la evolución hacia las formas más severas de disfunción cardíaca por enfermedad de Chagas. El potencial inmunomodulador del tratamiento con benznidazol durante la fase crónica podría ayudar a equilibrar los niveles de MIF y la patología inflamatoria en esta etapa de la infección, limitando así el riesgo de daño cardiovascular a largo plazo

Immunomodulatory and anti-fibrotic effects of ganglioside therapy on the cardiac chronic form of experimental Trypanosoma cruzi infection

Heart failure and sudden death are the most common causes of death in patients with Chagas' disease. The main drug available for Chagas treatment is benznidazole, which eradicates Trypanosoma cruzi parasites during the acute stage of infection. However, its effectiveness during the chronic phase remains unclear. Ganglioside GM1 administration in chronically infected patients resulted to be an effective treatment for the cardiac manifestations of Chagas' disease. However, the precise mechanisms of GM1-induced improvement during chronic T. cruzi infection still remain unknown. The aim of the present study was to evaluate the potential benefits of ganglioside GM1 treatment during the chronic stage of murine chagasic infection, analyzing its influence on myocardial pathology as well as its immunomodulatory effects. The results obtained showed that GM1 therapy diminished the extent of myocardial fibrosis induced by T. cruzi in chronically infected mice. In addition, GM1 treatment resulted in a significant reduction in the myocardial expression of the fibrogenic cytokine TGF-β as well as the proinflammatory cytokines and chemokines IFN-γ, TNF-α and CCL5/RANTES. Our experimental data indicate that GM1 could be a promising mmunomodulatory agent with capacity to limit the inflammatory process leading to myocardial tissue damage in chronic chagasic patients.Fil: Cutrullis, Romina Andrea. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Servicio de Parasitología y Chagas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Poklépovich Caride, Tomás Javier. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Servicio de Parasitología y Chagas; ArgentinaFil: Postan, Miriam. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Servicio de Parasitología y Chagas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Freilij, Hector León. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Servicio de Parasitología y Chagas; ArgentinaFil: Petray, Patricia Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Servicio de Parasitología y Chagas; Argentin

Archaeosomes made of Halorubrum tebenquichense total polar lipids: A new source of adjuvancy

Background: Archaeosomes (ARC), vesicles prepared from total polar lipids (TPL) extracted from selected genera and species from the Archaea domain, elicit both antibody and cell-mediated immunity to the entrapped antigen, as well as efficient cross priming of exogenous antigens, evoking a profound memory response. Screening for unexplored Archaea genus as new sources of adjuvancy, here we report the presence of two new Halorubrum tebenquichense strains isolated from grey crystals (GC) and black mood (BM) strata from a littoral Argentinean Patagonia salt flat. Cytotoxicity, intracellular transit and immune response induced by two subcutaneous (sc) administrations (days 0 and 21) with BSA entrapped in ARC made of TPL either form BM (ARC-BM) and from GC (ARC-GC) at 2% w/w (BSA/lipids), to C3H/HeN mice (25 μg BSA, 1.3 mg of archaeal lipids per mouse) and boosted on day 180 with 25 μg of bare BSA, were determined. Results: DNA G+C content (59.5 and 61.7% mol BM and GC, respectively), 16S rDNA sequentiation, DNA-DNA hybridization, arbitrarily primed fingerprint assay and biochemical data confirmed that BM and GC isolates were two non-previously described strains of H. tebenquichense. Both multilamellar ARC mean size were 564 ± 22 nm, with -50 mV zeta-potential, and were not cytotoxic on Vero cells up to 1 mg/ml and up to 0.1 mg/ml of lipids on J-774 macrophages (XTT method). ARC inner aqueous content remained inside the phago-lysosomal system of J-774 cells beyond the first incubation hour at 37°C, as revealed by pyranine loaded in ARC. Upon subcutaneous immunization of C3H/HeN mice, BSA entrapped in ARC-BM or ARC-GC elicited a strong and sustained primary antibody response, as well as improved specific humoral immunity after boosting with the bare antigen. Both IgG1 and IgG2a enhanced antibody titers could be demonstrated in long-term (200 days) recall suggesting induction of a mixed Th1/Th2 response. Conclusion: We herein report the finding of new H. tebenquichense non alkaliphilic strains in Argentinean Patagonia together with the adjuvant properties of ARC after sc administration in mice. Our results indicate that archaeosomes prepared with TPL from these two strains could be successfully used as vaccine delivery vehicles

Archaeosomes made of Halorubrum tebenquichense total polar lipids: a new source of adjuvancy

<p>Abstract</p> <p>Background</p> <p>Archaeosomes (ARC), vesicles prepared from total polar lipids (TPL) extracted from selected genera and species from the Archaea domain, elicit both antibody and cell-mediated immunity to the entrapped antigen, as well as efficient cross priming of exogenous antigens, evoking a profound memory response. Screening for unexplored Archaea genus as new sources of adjuvancy, here we report the presence of two new <it>Halorubrum tebenquichense </it>strains isolated from grey crystals (<it>GC</it>) and black mood (<it>BM</it>) strata from a littoral Argentinean Patagonia salt flat. Cytotoxicity, intracellular transit and immune response induced by two subcutaneous (sc) administrations (days 0 and 21) with BSA entrapped in ARC made of TPL either form <it>BM </it>(ARC-BM) and from <it>GC </it>(ARC-GC) at 2% w/w (BSA/lipids), to C3H/HeN mice (25 μg BSA, 1.3 mg of archaeal lipids per mouse) and boosted on day 180 with 25 μg of bare BSA, were determined.</p> <p>Results</p> <p>DNA G+C content (59.5 and 61.7% mol <it>BM </it>and <it>GC</it>, respectively), 16S rDNA sequentiation, DNA-DNA hybridization, arbitrarily primed fingerprint assay and biochemical data confirmed that <it>BM </it>and <it>GC </it>isolates were two non-previously described strains of <it>H. tebenquichense</it>. Both multilamellar ARC mean size were 564 ± 22 nm, with -50 mV zeta-potential, and were not cytotoxic on Vero cells up to 1 mg/ml and up to 0.1 mg/ml of lipids on J-774 macrophages (XTT method). ARC inner aqueous content remained inside the phago-lysosomal system of J-774 cells beyond the first incubation hour at 37°C, as revealed by pyranine loaded in ARC. Upon subcutaneous immunization of C3H/HeN mice, BSA entrapped in ARC-BM or ARC-GC elicited a strong and sustained primary antibody response, as well as improved specific humoral immunity after boosting with the bare antigen. Both IgG1 and IgG2a enhanced antibody titers could be demonstrated in long-term (200 days) recall suggesting induction of a mixed Th1/Th2 response.</p> <p>Conclusion</p> <p>We herein report the finding of new <it>H. tebenquichense </it>non alkaliphilic strains in Argentinean Patagonia together with the adjuvant properties of ARC after sc administration in mice. Our results indicate that archaeosomes prepared with TPL from these two strains could be successfully used as vaccine delivery vehicles.</p

Benznidazole Therapy Modulates Interferon-γ and M2 Muscarinic Receptor Autoantibody Responses in Trypanosoma cruzi-Infected Children

OBJECTIVE: The presence of autoantibodies with adrenergic and cholinergic activity, capable of triggering neurotransmitter receptor-mediated effects, has been associated with pathogenesis in T. cruzi-infected hosts. The goal of this study was to investigate the production of anti-M2 muscarinic receptor autoantibodies (Anti-M2R AAbs) as well as the IFN-γ profile in children at the early stage of Chagas disease, and to examine whether trypanocidal chemotherapy with benzonidazole (BZ) could modify both response patterns. METHODS: This study comprised 30 T. cruzi-infected children (mean age: 13.8 years) and 19 uninfected controls (mean age: 12.7 years). Infected patients were treated with BZ and followed-up. Blood samples collected at diagnosis-T0, end of treatment-T1, and six months later-T2 were analysed by ELISA for detection of Anti-M2R AAbs and circulating levels of IFN-γ. RESULTS: At T0, anti-M2R AAbs were demonstrated in 56.7% of T. cruzi-infected patients, whereas uninfected controls were 100% negative. The average age of Anti-M2R AAbs(+) patients was higher than that from negative population. Infected children also displayed significantly stronger serum IFN-γ responses than controls. Upon BZ treatment, a significant linear decreasing trend in Anti-M2R AAb reactivity was recorded throughout the follow-up, with 29.7-88.1% decrease at T2. IFN-γ circulating levels also declined by T2. CONCLUSION: Anti-M2R AAbs and IFN-γ raise early during chagasic infection in children and are downmodulated by BZ therapy. These findings reinforce the usefulness of early BZ treatment not only to eliminate the parasite but also to reduce potentially pathogenic immune responses

Polyfunctional T cell responses in children in early stages of chronic Trypanosoma cruzi infection contrast with monofunctional responses of long-term infected adults

Background: Adults with chronic Trypanosoma cruzi exhibit a poorly functional T cell compartment, characterized by monofunctional (IFN-γ-only secreting) parasite-specific T cells and increased levels of terminally differentiated T cells. It is possible that persistent infection and/or sustained exposure to parasites antigens may lead to a progressive loss of function of the immune T cells. Methodology/Principal Findings: To test this hypothesis, the quality and magnitude of T. cruzi-specific T cell responses were evaluated in T. cruzi-infected children and compared with long-term T. cruzi-infected adults with no evidence of heart failure. The phenotype of CD4+ T cells was also assessed in T. cruzi-infected children and uninfected controls. Simultaneous secretion of IFN-γ and IL-2 measured by ELISPOT assays in response to T. cruzi antigens was prevalent among T. cruzi-infected children. Flow cytometric analysis of co-expression profiles of CD4+ T cells with the ability to produce IFN-γ, TNF-α, or to express the co-stimulatory molecule CD154 in response to T. cruzi showed polyfunctional T cell responses in most T. cruzi-infected children. Monofunctional T cell responses and an absence of CD4+TNF-α+-secreting T cells were observed in T. cruzi-infected adults. A relatively high degree of activation and differentiation of CD4+ T cells was evident in T. cruzi-infected children. Conclusions/Significance: Our observations are compatible with our initial hypothesis that persistent T. cruzi infection promotes eventual exhaustion of immune system, which might contribute to disease progression in long-term infected subjects.Fil: Albareda, María Cecilia. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud. Instituto Nacional de Parasitología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Provincia de Buenos Aires. Ministerio de Salud. Hospital Interzonal de Agudos "Eva Perón"; ArgentinaFil: de Rissio, Ana María. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud. Instituto Nacional de Parasitología; ArgentinaFil: Tomas, Gonzalo. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud. Instituto Nacional de Parasitología; ArgentinaFil: Serjan, Alicia. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Juan A. Fernández"; ArgentinaFil: Alvarez, María Gabriela. Provincia de Buenos Aires. Ministerio de Salud. Hospital Interzonal de Agudos "Eva Perón"; ArgentinaFil: Viotti, Rodolfo Jorge. Provincia de Buenos Aires. Ministerio de Salud. Hospital Interzonal de Agudos "Eva Perón"; ArgentinaFil: Fichera, Laura Edith. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud. Instituto Nacional de Parasitología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Esteva, Mónica Inés. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud. Instituto Nacional de Parasitología; ArgentinaFil: Potente, Daniel Fernando. Provincia de Buenos Aires. Ministerio de Salud. Hospital Interzonal de Agudos "Eva Perón"; ArgentinaFil: Armenti, Alejandro. Provincia de Buenos Aires. Ministerio de Salud. Hospital Interzonal de Agudos "Eva Perón"; ArgentinaFil: Tarleton, Rick L.. University of Georgia; Estados UnidosFil: Laucella, Susana Adriana. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud. Instituto Nacional de Parasitología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Archaeosomes made of Halorubrum tebenquichense total polar lipids: A new source of adjuvancy

Background: Archaeosomes (ARC), vesicles prepared from total polar lipids (TPL) extracted from selected genera and species from the Archaea domain, elicit both antibody and cell-mediated immunity to the entrapped antigen, as well as efficient cross priming of exogenous antigens, evoking a profound memory response. Screening for unexplored Archaea genus as new sources of adjuvancy, here we report the presence of two new Halorubrum tebenquichense strains isolated from grey crystals (GC) and black mood (BM) strata from a littoral Argentinean Patagonia salt flat. Cytotoxicity, intracellular transit and immune response induced by two subcutaneous (sc) administrations (days 0 and 21) with BSA entrapped in ARC made of TPL either form BM (ARC-BM) and from GC (ARC-GC) at 2% w/w (BSA/lipids), to C3H/HeN mice (25 μg BSA, 1.3 mg of archaeal lipids per mouse) and boosted on day 180 with 25 μg of bare BSA, were determined. Results: DNA G+C content (59.5 and 61.7% mol BM and GC, respectively), 16S rDNA sequentiation, DNA-DNA hybridization, arbitrarily primed fingerprint assay and biochemical data confirmed that BM and GC isolates were two non-previously described strains of H. tebenquichense. Both multilamellar ARC mean size were 564 ± 22 nm, with -50 mV zeta-potential, and were not cytotoxic on Vero cells up to 1 mg/ml and up to 0.1 mg/ml of lipids on J-774 macrophages (XTT method). ARC inner aqueous content remained inside the phago-lysosomal system of J-774 cells beyond the first incubation hour at 37°C, as revealed by pyranine loaded in ARC. Upon subcutaneous immunization of C3H/HeN mice, BSA entrapped in ARC-BM or ARC-GC elicited a strong and sustained primary antibody response, as well as improved specific humoral immunity after boosting with the bare antigen. Both IgG1 and IgG2a enhanced antibody titers could be demonstrated in long-term (200 days) recall suggesting induction of a mixed Th1/Th2 response. Conclusion: We herein report the finding of new H. tebenquichense non alkaliphilic strains in Argentinean Patagonia together with the adjuvant properties of ARC after sc administration in mice. Our results indicate that archaeosomes prepared with TPL from these two strains could be successfully used as vaccine delivery vehicles.Facultad de Ciencias Exacta

ADENOSINE DEAMINASE ACTIVITY AND SERUM C-REACTIVE PROTEIN AS PROGNOSTIC MARKERS OF CHAGAS DISEASE SEVERITY

SUMMARY Chagas disease is a public health problem worldwide. The availability of diagnostic tools to predict the development of chronic Chagas cardiomyopathy is crucial to reduce morbidity and mortality. Here we analyze the prognostic value of adenosine deaminase serum activity (ADA) and C-reactive protein serum levels (CRP) in chagasic individuals. One hundred and ten individuals, 28 healthy and 82 chagasic patients were divided according to disease severity in phase I (n = 35), II (n = 29), and III (n = 18). A complete medical history, 12-lead electrocardiogram, chest X-ray, and M-mode echocardiogram were performed on each individual. Diagnosis of Chagas disease was confirmed by ELISA and MABA using recombinant antigens; ADA was determined spectrophotometrically and CRP by ELISA. The results have shown that CRP and ADA increased linearly in relation to disease phase, CRP being significantly higher in phase III and ADA at all phases. Also, CRP and ADA were positively correlated with echocardiographic parameters of cardiac remodeling and with electrocardiographic abnormalities, and negatively with ejection fraction. CRP and ADA were higher in patients with cardiothoracic index ≥ 50%, while ADA was higher in patients with ventricular repolarization disturbances. Finally, CRP was positively correlated with ADA. In conclusion, ADA and CRP are prognostic markers of cardiac dysfunction and remodeling in Chagas disease

MIF-driven activation of macrophages induces killing of intracellular Trypanosoma cruzi dependent on endogenous production of tumor necrosis factor, nitric oxide and reactive oxygen species

The proinflammatory cytokine macrophage migration inhibitory factor (MIF) is a key player in innate immunity. MIF has been considered critical for controlling acute infection by the protozoan Trypanosoma cruzi, but the underlying mechanisms are poorly understood. Our study aimed to analyze whether MIF could favor microbicidal activity of the macrophage, a site where T. cruzi grows and the initial effector cell against this parasite. Using murine macrophages infected in vitro, we examined the effect of MIF on their parasiticidal ability and attempted to identify inflammatory agents involved in MIF-induced protection. Our findings show that MIF is readily secreted from peritoneal macrophages upon T. cruzi infection. MIF activates both primary and J774 phagocytes boosting the endogenous production of tumor necrosis factor-alpha via mitogen-activated protein kinase p38 signaling, as well as the release of nitric oxide and reactive oxygen species, leading to enhanced pathogen elimination. MIF can also potentiate the effect of interferon-gamma on T. cruzi killing by J774 and mouse peritoneal macrophages, rendering these cells more competent in reducing intracellular parasite burden. The present results unveil a novel innate immune pathway that contributes to host defense and broaden our understanding of the regulation of inflammatory mediators implicated in early parasite containment that is decisive for resistance to T. cruzi infection.Fil: Cutrullis, Romina Andrea. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños ; ArgentinaFil: Petray, Patricia Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Corral, Ricardo Santiago. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños ; Argentin

Timing of expression of inflammatory mediators in skeletal muscles from mice acutely infected with the RA strain of Trypanosoma cruzi

Fil: Cutrullis, Romina Andrea. Servicio de Parasitología y Chagas, Hospital de Niños Dr. Ricardo Gutiérrez; Argentina.Fil: Postan, Miriam. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología; Argentina.Fil: Petray, Patricia Beatriz. Servicio de Parasitología y Chagas, Hospital de Niños Dr. Ricardo Gutiérrez; Argentina.Fil: Corral, Ricardo Santiago. Servicio de Parasitología y Chagas, Hospital de Niños Dr. Ricardo Gutiérrez; Argentina.Chagas' disease is caused by persistent Trypanosoma cruzi infection in muscle cells that ultimately results in chronic inflammation and tissue destruction. The goal of this study was to determine the expression of different chemokines and their receptors, as well as proinflammatory cytokines and inducible nitric oxide synthase, in muscles from mice acutely infected with T. cruzi