Recombinant antibody against Trypanosoma cruzi from patients with chronic Chagas heart disease recognizes mammalian nervous system.

Background: To deeply understand the role of antibodies in the context of Trypanosoma cruzi infection, we decided to characterize A2R1, a parasite antibody selected from single-chain variable fragment (scFv) phage display libraries constructed from B cells of chronic Chagas heart disease patients. Methods: Immunoblot, ELISA, cytometry, immunofluorescence and immunohistochemical assays were used to characterize A2R1 reactivity. To identify the antibody target, we performed an immunoprecipitation and two-dimensional electrophoresis coupled to mass spectrometry and confirmed A2R1 specific interaction by producing the antigen in different expression systems. Based on these data, we carried out a comparative in silico analysis of the protein target_s orthologues, focusing mainly on post-translational modifications. Findings: A2R1 recognizes a parasite protein of ~50 kDa present in all life cycle stages of T. cruzi, as well as in other members of the kinetoplastid family, showing a defined immunofluorescence labeling pattern consistent with the cytoskeleton. A2R1 binds to tubulin, but this interaction relies on its post-translational modifications. Interestingly, this antibody also targets mammalian tubulin only present in brain, staining in and around cell bodies of the human peripheral and central nervous system. Interpretation: Our findings demonstrate for the first time the existence of a human antibody against T. cruzi tubulin capable of cross-reacting with a human neural protein. This work re-emphasizes the role of molecular mimicry between host and parasitic antigens in the development of pathological manifestations of T. cruzi infection

DNA immunization with the ribosomal P2beta gene of Trypanosoma cruzi fails to induce pathogenic antibodies

Patients with chronic Chagas' heart disease (cChHD) develop a strong IgG response against the C-terminal region of the Trypanosoma cruzi ribosomal P2beta protein (TcP2beta). These antibodies have been shown to exert an in vitro chronotropic effect on cardiocytes through stimulation of the beta1-adrenergic receptor (beta1-AR). Moreover, the presence of antibodies recognizing the TcP2beta C-terminus was associated with cardiac alterations in mice immunized with the corresponding recombinant protein. Here, we demonstrate that DNA immunization could be used to modulate the specificity of the anti-TcP2beta humoral response in order to avoid the production of pathogenic antibodies. After DNA injection, we detected IgG antibodies that were directed only to internal epitopes of the TcP2beta molecule and that did not exert anti-beta1-AR functional activity, measured as an increase in intracellular cAMP levels of transfected COS-7 cells. Accordingly, DNA-immunized mice did not present electrocardiographic alterations. These data demonstrate that anti-TcP2beta antibodies elicited by DNA immunization are completely different in their specificity and functional activity from those produced during T. cruzi infectionFil: Levitus, Gabriela Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular ; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular. Laboratorio de Fisiología y Biología Molecular; ArgentinaFil: Grippo, Vanina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular. Laboratorio de Fisiología y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular ; ArgentinaFil: Labovsky, Vivian. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular. Laboratorio de Fisiología y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular ; ArgentinaFil: Abascal, Sergio. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular. Laboratorio de Fisiología y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular ; ArgentinaFil: Hontebeyrie, Mireille. Instituto Pasteur. Departement de Biologie Structurale Et Chimie; FranciaFil: Levin, Mariano Jorge. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular. Laboratorio de Fisiología y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular ; Argentin

Resultados de un estudio multicéntrico, no controlado, de seguimiento sobre factores de riesgo cardiovascular

Background Cardiovascular disease is still one of the leading causes of mortality in our country. It has been well documented that adequate changes in lifestyle are beneficial for primary and secondary prevention. However, it is difficult to apply or maintain these measures in the real world. The ENASE study was developed as a program to train physicians in the knowledge of good dietary habits and exercise to help patients to incorporate healthy behaviors in everyday life. Objective To evaluate if a plan designed to train attending physicians can produce changes in the patients. Material and Methods We conducted a prospective, multicenter and uncontrolled study on 508 patients with a follow-up of three years in 17 provinces and in the Autonomous City of Buenos Aires. Statistical analysis was performed using the paired Student�s t test and the Wilcoxon test. Results With the help of primary physicians, this population increased physical activity and made significant changes in eating habits. Blood pressure, triglycerides, LDL-cholesterol and blood sugar levels decreased significantly and progressively from the first to the third year of follow-up. HDL-cholesterol increased significantly while body weight showed a slight and non-significant reduction. The prescription of medical treatment did not change during the three years, except for a greater use of statins in accordance with the recommendations suggested by national guidelines. Conclusions This study demonstrates that a plan focused on educating and training physicians produces significant benefits on the cardiovascular health of patients.Introducción La enfermedad cardiovascular sigue siendo una de las principales causas de muerte en nuestro país y está bien documentado que cambios adecuados en el estilo de vida son favorables tanto en términos de prevención primaria como secundaria. Sin embargo, en el mundo real, estas medidas no son de fácil instrumentación o mantenimiento. Esto llevó al desarrollo del estudio ENASE, que se concibió como un programa de entrenamiento del médico en el conocimiento de buenos hábitos de alimentación y ejercicio y en cómo ayudar al paciente a que pueda incorporarlos en forma metódica en la vida cotidiana. Objetivo Evaluar si un plan de educación del médico asistencial puede producir cambios en los pacientes. Material y métodos Estudio prospectivo, no controlado, multicéntrico, con seguimiento a 3 años en 17 provincias y en la Ciudad Autónoma de Buenos Aires, que incluyó 508 pacientes. Para el análisis estadístico se emplearon la prueba de la t de Student para muestras apareadas y la prueba de Wilcoxon. Resultados Con la guía del médico de cabecera, esta población en estudio incrementó la actividad física y realizó cambios significativos en la alimentación. A partir del primer año y con progreso adicional hasta los 3 años, se observó una reducción significativa de la presión arterial, los triglicéridos, el colesterol LDL y de la glucemia respecto de los datos basales. El colesterol HDL se incrementó en forma significativa, en tanto que el peso sólo mostró un leve descenso, no significativo. No hubo cambios en la prescripción de fármacos durante los 3 años, con excepción de un incremento en el uso de estatinas, acorde al mayor espectro de indicaciones sugeridas por las normativas nacionales. Conclusiones Este estudio demuestra que un plan de educación y entrenamiento del médico para actuar en el paciente produce beneficios significativos en la salud cardiovascular

The heavy chain variable segment gene repertoire in chronic Chagas' heart disease

Patients chronically infected with Trypanosoma cruzi develop chronic Chagas' heart disease (cChHD). Their Ab response is suspected to be involved in the cardiac pathogenesis. Reactivity of serum Abs from these patients has been extensively studied but little is known about the diversity of the in vivo IgG repertoire. We analyzed 125 variable H chain (VH) genes and compared it to repertoires from healthy individuals, and patients with autoimmune processes and other infections. VH were from plasma cells isolated from heart tissue of three cChHD patients and from a Fab combinatorial library derived from bone marrow of another cChHD patient. The role of the parasite in shaping the Ab repertoire was assessed analyzing VH genes before and after panning against T. cruzi Ag. Among recovered VH genes, a significantly increased representation of VH4 was observed. Plasma cells at the site of cardiac infiltration showed an increased VH1 usage. CDR3 lengths were similar to the ones found in the healthy repertoire and significantly shorter than in other infections. VH derived from anti-T. cruzi Fab and plasma cells showed a higher proportion of hypermutated genes, 46.9% and 43.75%, respectively, vs 30.9% of the cChHD patient repertoire, pointing to the role of parasite Ags in the shaping of the humoral response in Chagas' disease. No histological evidence of germinal center-like structures was observed in heart tissue. In accordance, VH analysis of heart plasmocytes revealed no evidence of clonal B cell expansion, suggesting that they migrated into heart tissue from secondary lymphoid organs.Fil: Grippo, Vanina. 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; ArgentinaFil: Mahler, Evelyn. 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; ArgentinaFil: Elias, Fernando E.. Deutsches Rheumaforschungszentrum; AlemaniaFil: Cauerhff, Ana. 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; ArgentinaFil: 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; ArgentinaFil: Tentori, Maria C.. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Juan A. Fernández"; ArgentinaFil: Ruiz, Aurora. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Juan A. Fernández"; ArgentinaFil: Vigliano, Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación Favaloro; ArgentinaFil: Laguens, Rubén. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación Favaloro; ArgentinaFil: Berek, Claudia. Deutsches Rheumaforschungszentrum; AlemaniaFil: Levin, Mariano Jorge. 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; Argentin

Serological based monitoring of a cohort of patients with chronic Chagas disease treated with benznidazole in a highly endemic area of northern Argentina

This study aimed to evaluate well-documented diagnostic antigens, named B13, 1F8 and JL7 recombinant proteins, as potential markers of seroconversion in treated chagasic patients. Prospective study, involving 203 patients treated with benznidazole, was conducted from endemic areas of northern Argentina. Follow-up was possible in 107 out of them and blood samples were taken for serology and PCR assays before and 2, 3, 6, 12, 24 and 36 months after treatment initiation. Reactivity against Trypanosoma cruzi lysate and recombinant antigens was measured by ELISA. The rate of decrease of antibody titers showed nonlinear kinetics with an abrupt drop within the first three months after initiation of treatment for all studied antigens, followed by a plateau displaying a low decay until the end of follow-up. At this point, anti-B13, anti-1F8 and anti-JL7 titers were relatively close to the cut-off line, while anti-T. cruzi antibodies still remained positive. At baseline, 60.8% (45/74) of analysed patients tested positive for parasite DNA by PCR and during the follow-up period in 34 out of 45 positive samples (75.5%) could not be detected T. cruzi DNA. Our results suggest that these antigens might be useful as early markers for monitoring antiparasitic treatment in chronic Chagas disease

VHH mutation alters affinity, potency and breadth of neutralization.

<p>(A) VHH binding to clade B gp120 Bal.26 in ELISA detected by their C-terminal Myc tag. (B) VHH neutralization of Bal.26 HIV pseudovirus in the TZM-bl assay. Mutant VHH were generated by site-directed mutagenesis as detailed in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004552#s4" target="_blank">Materials and Methods</a>. (C) The fold change in IC50 µg/ml values for B9 S54W relative to B9, indicating the increase potency of the mutant are plotted on the Y-axis. The IC50 µg/ml values for B9 against each virus in the legend are plotted on the X-axis, indicating the baseline potency of B9. All assays were carried out in duplicate and error bars represent standard deviation. These data are representative of at least three independent experiments.</p

A14, B9, B21 binding to HIV ENV.

<p>VHH binding to immunogens (A) clade B gp140 R2, (B) clade C gp140 96ZM651.02, (C) clade A 92UG037 gp140, (D) CRF BC CN54 gp140, (E) YU2 WT and D368R gp120, and (F) RSC3 mutant and RSC3 delta mutant gp120, was assessed by ELISA as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004552#s4" target="_blank">Materials and Methods</a>. The positive control for gp140 binding was J3 (McCoy et al. 2012) that for gp41 binding 2H10 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004552#ppat.1004552-LutjeHulsik1" target="_blank">[32]</a>. The positive control for both RSC3 gp120 proteins was D47 a non-neutralizing RSC3-specific VHH (L McCoy unpublished data). All binding assays were carried out in duplicate and error bars represent standard deviation. These data are representative of at least three independent experiments.</p