Induction of antibodies reactive to cardiac myosin and development of heart alterations in cruzipain-immunized mice and their offspring.

Human and murine infection with Trypanosoma cruzi parasite is usually accompanied by strong humoral and cellular immune response to cruzipain, a parasite immunodominant antigen. In the present study we report that the immunization of mice with cruzipain devoid of enzymatic activity, was able to induce antibodies which bind to a 223-kDa antigen from a mouse heart extract. We identified this protein as the mouse cardiac myosin heavy chain by sequencing analysis. The study of IgG isotype profile revealed the occurrence of all IgG isotypes against cruzipain and myosin. IgG1 showed the strongest reactivity against cruzipain, whereas IgG2a was the main isotype against myosin. Anti-cruzipain antibodies purified by immunoabsorption recognized the cardiac myosin heavy chain, suggesting cross-reactive epitopes between cruzipain and myosin. Autoimmune response in mice immunized with cruzipain was associated to heart conduction disturbances. In addition, ultrastructural findings revealed severe alterations of cardiomyocytes and IgG deposit on heart tissue of immunized mice. We investigated whether antibodies induced by cruzipain transferred from immunized mothers to their offsprings could alter the heart function in the pups. All IgG isotypes against cruzipain derived from transplacental crossing were detected in pups' sera. Electrocardiographic studies performed in the offsprings born to immunized mothers revealed conduction abnormalities. These results provide strong evidence for a pathogenic role of autoimmune response induced by a purified T. cruzi antigen in the development of experimental Chagas' disease.

Trypanosoma cruzi: immune response and functional heart damage induced in mice by the main linear B-cell epitope of parasite ribosomal P proteins.

We report herein on the specific and autoimmune humoral response generated by the immunization of mice with the R13 synthetic peptide coupled to a carrier protein, OVA. This peptide corresponds to the C-terminal region of Trypanosoma cruzi ribosomal P1 and P2 proteins (EEEDDDMGFGLFD), a sequence that differs from the other eukariotic P consensus sequence (EESDDDMGFGLFD) only in a nonconservative amino acid substitution. The antibody response studied by ELISA revealed that all R13-immunized mice had antibodies against R13, consisting mainly of IgG1 and IgG2 isotypes, even though IgG3 and IgE isotypes were also observed. The self-reactivity of anti-R13 sera assayed by immunoblot, revealed that all sera contained IgG antibodies binding to mouse and human 38-kDa heart antigen. This antigenic band binds several immunoglobulin isotypes (IgG2 > IgG3 > IgE > IgG1). The specificity of anti-R13 antibodies analyzed by competitive inhibition of R13 ELISA using R13 and R7 (MGFGLFD) peptides revealed that the reactivity of the induced anti-P antibodies was not absorbed by R7. Therefore, the main immunogenic region of R13 for mouse would be EEEDDD, which contains the amino acid substitution. In parallel with this humoral response, both partial protection and heart damage were observed in R13-immunized mice. In fact, the R13-immunized mice showed significantly lower parasitemia and longer survival than the control animals. In addition, all R13-immunized mice showed electrocardiographic changes (bradycardia, prolonged PQ segment, and intraventricular conduction disturbances), which are typical findings in Chagas disease patients. This study represents the first definitive report in which one defined B-cell epitope, the single peptide R13 from T. cruzi, coupled to a carrier protein was able to induce specific and autoreactive antibodies as well as to generate heart functional alterations.

Anomalous intraocular pressure changes in Chagas' disease elicited by postural test.

We compared the effects of postural changes on intraocular pressure, systemic blood pressure, and pupil size with and without induced mydriasis in 15 chronic chagasic patients and 20 healthy age-matched controls. The chagasic patients showed a marked fall in intraocular pressure on rising. However, systemic systolic blood pressure changes and pupil size in patients did not differ from those measured in controls. Our findings may be explained by an alteration in the autonomic ocular system that regulates homeostasis of ocular pressure and the probable existence of a baroreceptor arc-reflex that restores the equilibrium of sudden changes in the intraocular pressure.

Chagas' disease: polyspecificity of antibodies against Trypanosoma cruzi acidic antigens.

The aim of this work was to study the reactivity of chagasic patient sera against a panel of natural antigens and its relationship with the immune response against T. cruzi acidic antigens. The presence of IgG and IgM antibodies reactive with myosin, myoglobin, actin and thyroglobulin was investigated in sera with positive serology for Chagas' disease classified into groups (G) I, n = 7, with normal electrocardiogram (ECG) and no signs or symptoms of the disease; GII, n = 7, with ECG abnormalities but without cardiomegaly and GIII, n = 7, with cardiomegaly and congestive heart failure. Healthy individual sera were analyzed in parallel as controls. In the three groups of chagasic patients, a high proportion of sera exhibited an enhancement of IgG response anti actin ranging from 71 to 100%. IgM against this antigen was found positive in GI, 21%; GII and GIII, 57%. The antibodies binding to myosin and myoglobin were mainly of IgM type. When myosin was assayed, the frequency of reactive sera was gradually diminished as heart involvement increased: GI 57%, GII 28% and GIII 14%. Only IgG antibodies against thyroglobulin were detected in the three groups of chagasic patients ranging from 43 to 86%. IgG natural antibodies showed to be polyreactive, since a diminished reactivity against each one of the natural antigens assayed and against T. cruzi acidic antigens (FIV) was observed in the sera absorbed with any of the selected antigens irrespective of the absorbing ones. Moreover, the antibodies against FIV parasite's antigens purified by immunoabsorption showed a similar reactivity with FIV, myosin and actin, and a slight lower reactivity with thyroglobulin. These results indicate that in chagasic patients, the specific humoral response against FIV is associated with an increase of the natural autoantibodies along with their polyspecificity.

Chronic Chagas' disease: possible mechanism of sinus bradycardia.

OBJECTIVE: To determine the cause of sinus bradycardia in patients with chronic Chagas' disease by studying their sinus node function with pharmacological agents. DESIGN: Cohort study. SETTING: Referral tertiary care hospital in Cordoba, Argentina. PATIENTS: Thirty-seven chagasic patients (19 females and 18 males, mean age 41 +/- 12 years) were divided into three groups: group 1, 14 patients with positive serology for Chagas' disease but no evidence of cardiac involvement; group 2, 14 patients with Chagas' disease and electrocardiographic abnormalities but without signs or symptoms of congestive heart failure or cardiomegaly; and group 3, nine patients with Chagas' disease and evidence of cardiomegaly and congestive heart failure. Six healthy volunteers with no evidence of heart disease were used as controls. INTERVENTIONS: All subjects received 0.04 mg/kg intravenous atropine sulphate followed 3 mins later by 0.2 mg/kg intravenous propranolol slowly injected. The resultant heart rate obtained with this total autonomic blockade (TAB) was considered to be the intrinsic heart rate observed (IHRo). MAIN RESULTS: The mean increase of heart rate after atropine was +68 beats/min in controls, +45 beats/min in group 1, +45 beats/min in group 2 and +32 beats/min in group 3. The change from the basal heart rate to the IHRo after TAB with atropine and propranolol was +29 beats/min in controls, +17 beats/min in group 1, +17 beats/min in group 2 and +5 beats/min in group 3. CONCLUSIONS: The observed response to atropine in patients with Chagas' disease suggests abnormality in the innervation of the sinus node. The difference found in the IHRo after TAB indicates involvement of the automaticity of the sinus node. These findings may explain the bradycardia reported in this disease.

La disfunción del sistema nervioso autónomo en la enfermedad de chagas en Venezuela / Autonomic nervous system dysfunction in the chagas diseases in Venezuela

1.-Se estudio una población con Enfermedad de Chagas en el medio venezolano, integrada por 181 personas. Las pruebas clínico-fisiológicas permitieron demostrar anormalidades de los reflejos cardiovasculares y por consiguiente permitieron establecer la existencia de disfunción del sistema nervioso autónomo en la Enfermedad de Chagas en el medio venezolano. 2.-La frecuencia de la disfunción autonómica en esta serie fue elevada, del orden del 54 por ciento (97/181). La disfunción puede presentarse en las tres etapas evolutivas de la afección pero aparece con frecuencia en la etapa inicial, en pacientes jóvenes, asintomáticos y puede constituir la disfunción autonómica en este subgrupo la primera y única evidencia de la enfermedad y no puede ser relacionada ni con la edad ni con la insuficiencia cardiaca

Ensayo terapéutico con gangliosidos en la cardioneuropatía chagasica en dos poblaciones sudamericas (Venezuela y Arganetina) / Therapeutic essay with gangliosides in the chagasic cardioneurophathy in two sudamerican population (Venezuela and Argentina)

Hasta el presente no se ha encontrado un tratamiento farmacológico efetivo de la cardioneuropatía chagásica, una de las causas más frecuentes de Insuficiencia Cardíaca Congestiva y de Muerte Súbita en el mundo. Se realizó un estudio en una población de 128 adultos con serología positiva para la Enfermedad de Chagas y pruebas anormales del Sistema Nervioso Autónomo. El estudio tuvo las características de: doble ciego paralelo y placebo-controlado y se ajustó a las normas, de la FDA; requeridas para la investigación en la aplicación de una nueva droga. Se utilizó Cronassial (Mezcla de Gangliósidos) en inyecciones intramuscular por 4 a 8 semanas. Se tomaron todas las normas de seguridad durante el tratamiento y 31 casos fueron excluídos por presentar Insuficiencia Cardíaca Congestiva. En los 97 pacientes restantes se determinó: la respuesta postural, mediante los cambios de la frecuencia cardíaca, presión arterial sistólica y del doble producto como respuesta al adoptar la posición erecta; del mismo modo, los cambios en la frecuencia cardíaca inducidos por el reflejo de la tos y de la hiperventilación. El Cronassial se mostró seguro y mediante el analisis estadístico ANOVA demostró que hubo mejoría significativa de la presión sistólica (P<0.14) y del doble producto (P<0.059) al estrés postural y de la respuesta de la frecuencia cardíaca a la Hiperventilación (0.017) comparado con el placebo

Chagas' disease cardioneuropathy: association of anti-Trypanosoma cruzi and anti-sciatic nerve antibodies.

The aim of this work was to study whether Trypanosoma cruzi infection could elicit humoral immune response to the well-defined parasite antigen acidic fraction separated from T. cruzi cytosol by isoelectric focusing and designated fraction IV (FIV) and whether this response could account for some of the autoreactive immune response against peripheral nerve components. Chagasic patients with positive serology for Chagas' disease were classified as group I (n = 12) with normal electrocardiograms (ECG) and no signs of disease, group II (n = 12) with ECG abnormalities but without cardiomegaly, and group III (n = 12) with cardiomegaly and congestive heart failure. Sera from patients in group II showed the highest frequency of positive reactivity against FIV. Ninety-two percent had titers higher than 1/400 while the percentage for groups I and III was 50%. The autoreactive response against human sciatic nerve saline extract (SNS) was studied. The binding of IgG to SNS was positive in groups I (58%), II (66%), and III (75%) patients. The treatment of SNS with periodate diminished the ability of antigens to fix IgG from these chagasic patients. Absorption studies were performed to investigate whether FIV and SNS could have cross-reactive epitopes. Preabsorption of positive sera with FIV inhibited 48-69% of samples' reactivity against antigen. In contrast, preabsorption of positive sera with SNS inhibited only 12-23% of samples' reactivity against antigen. Overall, these results suggest that FIV-T. cruzi and sciatic nerve components possess some epitopes, possibly of a carbohydrate nature, in common. Thus, infection in Chagas' disease could overcome the tolerance to self components and could lead to autoimmunity.

Chagas' cardioneuropathy: effect of ganglioside treatment in chronic dysautonomic patients--a randomized, double-blind, parallel, placebo-controlled study.

To date, there is no effective pharmacologic treatment for Chagas' cardioneuropathy, one of the most common causes of congestive heart failure and sudden death in the world. Fifty-eight adults with positive serology for Chagas' disease and abnormal autonomic nervous system tests participated in this placebo-controlled clinical trial with Cronassial (mixed gangliosides), 40 mg daily intramuscular injection for 4 or 8 weeks. We measured postural response (heart rate, systolic and diastolic arterial blood pressure changes in response to standing); heart rate changes induced by cough and hyperventilation reflex tests; dizziness on standing; number of stress-induced arrhythmias; and periodic acid-Schiff (PAS)-positive T-lymphocyte percentage in blood samples. Cronassial is safe and significantly improves systolic blood pressure (p = 0.050) and double product responses to postural stress (p = 0.028), hyperventilation heart rate response (p = 0.007), frequency of dizziness episodes (p less than 0.001), number of arrhythmias (p = 0.033), and percentage of PAS-positive T-lymphocyte counts (p less than 0.001) compared with placebo.

Impaired heart rate variability in patients with chronic Chagas' disease.

Spectral analysis of heart rate variability was used to investigate the possible alteration in sympathovagal control of heart rate in patients with Chagas' disease. The study included 31 subjects, divided into three groups: controls, Chagas' 1 (subjects with only positive serology for Chagas' disease) and Chagas' 2 (subjects with positive serology and electrocardiographic abnormalities that are usually found in this disease). The subjects were studied during rest, while standing, and during handgrip exercise. With this approach, the low frequency (approximately 0.1 Hz) spectral component of R-R interval variability is considered to be a marker primarily of sympathetic activity, whereas the high frequency (approximately 0.25 Hz) component, which is related to respiration, seems mainly to reflect vagal activity. We observed significant (p less than 0.05) differences among the three groups during standing: although in the control subjects the low-frequency component increased (delta = 30 +/- 5 normalized units, nu), there was no increase in Chagas' 1 (delta = -1 +/- 8 nu) and Chagas' 2 (delta = -2 +/- 8 nu) patients. During handgrip exercise, another test that is capable of exciting sympathetic outflow, there was an increase of low frequency only in control subjects. These results confirm the occurrence of quantitative and assessable abnormalities in the neural control of heart rate variability in Argentinian patients with chronic Chagas' disease, even in the absence of heart failure.

Plasma norepinephrine in Chagas' cardioneuromyopathy: a marker of progressive dysautonomia.

Chronic Chagas' disease produces pathologic changes of the cardiovascular, digestive, and autonomic nervous systems. In an attempt to elucidate the nature of the dysautonomia in patients with Chagas' disease, we measured plasma norepinephrine levels, blood pressure, and heart rate, both supine and standing in 26 patients, and compared these values of patients classified according to three clinical subsets of cardiovascular manifestations with the values of nine normal volunteers and 16 patients with nonchagasic heart failure. Results suggested (1) progressive blockade of the alpha receptor in patients with Chagas' disease who have minimal clinical symptoms (group I) and in those who have ECG alterations without congestive symptoms (group II), as reflected by normal or raised plasma norepinephrine levels without change of diastolic blood pressure during standing, which indicates absent postural reflexes; and (2) blockade associated with partial denervation in patients with Chagas' disease who have class III or IV heart failure (group III), as suggested by a lower supine plasma norepinephrine level and a fall in diastolic blood pressure in the upright position. The findings of reduced plasma norepinephrine levels are in contrast to the elevated plasma norepinephrine levels in patients without Chagas' disease with class III and IV heart failure who have sympathetic hyperactivity.