Cross-reactivity of antibodies in human infections by the kinetoplastid protozoa Trypanosoma cruzi, Leishmania chagasi and Leishmania (Viannia) braziliensis

We have detected antibodies, in the sera of Chagas disease, Kala-azar and Mucocutaneous leishmaniasis patients, that bind multiple antigens shared between the three causative agents. The Chagas disease sera showed 98 to 100% positive results by ELISA when the Leishmania braziliensis and Leishmania chagasi antigens were used, respectively. The Kala-azar sera showed 100% positive results with Trypanosoma cruzi or L. braziliensis antigens by immunofluorescence assays. The antibodies in the sera of Mucocutaneous leishmaniasis patients showed 100% positive results by ELISA assays with T. cruzi or L. chagasi antigens. Furthermore, the direct agglutination of L. chagasi promastigotes showed that 95% of Kala-azar and 35% of Mucocutaneous leishmaniasis sera agglutinated the parasite in dilutions above 1:512. In contrast, 15% of Chagas sera agglutinated the parasite in dilutions 1:16 and below. Western blot analysis showed that the Chagas sera that formed at least 24 bands with the T. cruzi also formed 13 bands with the L. chagasi and 17 bands with the L. braziliensis. The Kala-azar sera that recognized at least 29 bands with the homologous antigen also formed 14 bands with the T. cruzi and 10 bands with the L. braziliensis antigens. Finally, the Mucocutaneous leishmaniasis sera that formed at least 17 bands with the homologous antigen also formed 10 bands with the T. cruzi and four bands with the L. chagasi antigens. These results indicate the presence of common antigenic determinants in several protozoal proteins and, therefore, explain the serologic cross-reactions reported here.

Immunodiagnosis of chronic Chagas' disease using the Tc 46 and Tc 58 antigens

The polypeptides of 46 and 58kDa were recognized in different T. cruzi strains (Y, WSL and Colombiana) by serum of all chagasic patients studied. These polypeptides were isolated from T. cruzi Y strain and used in ELISA. The sensitivity and specificity were 97.6% [CI 95%: 86-100%] and 100% [CI 95%: 89.3-100%], respectively when Tc 46 was used. When Tc 58 was used the sensitivity and specificity were 100% [CI 95%: 89.6-100%] and 90.2% [CI 95%: 75.9-96.8%], respectively.

Trypanosoma cruzi in marsupial didelphids (Philander frenata and Didelhis marsupialis): differences in the humoral immune response in natural and experimental infections

Philander frenata and Didelphis marsupialis harbor parasitism by Trypanosoma cruzi without developing any apparent disease and on the contrary to D. marsupialis, P. frenata maintains parasitism by T. cruzi II subpopulations. Here we compared the humoral immune response of the two didelphids naturally and experimentally infected with T. cruzi II group, employing SDS-PAGE/Western blot techniques and by an Indirect immunofluorescence assay. We also studied the histopathological pattern of naturally and experimentally infected P. frenata with T. cruzi. P. frenata sera recognized more antigens than D. marsupialis, and the recognition pattern did not show any change over the course of the follow up of both didelphid species. Polypeptides of 66 and 90kDa were the most prominent antigens recognized by both species in the soluble and enriched membrane fractions. P. frenata recognized intensely also a 45kDa antigen. Our findings indicate that: 1) there were no quantitative or qualitative differences in the patent or subpatent phases in the recognition pattern of P. frenata; 2) the significant differences in the recognition pattern of parasitic antigens by P. frenata and D. marsupialis sera suggest that they probably "learned" to live in harmony with T. cruzi by different strategies; 3) although P. frenata do not display apparent disease, tissular lesions tended to be more severe than has been described in D. marsupialis; and 4) Both didelphids probably acquired infection by T. cruzi after their evolutionary divergence.

PARTICIPACIÓN DEL RECONOCIMIENTO INMUNE INNATO EN LA RESPUESTA DE CARDIOMIOCITOS DURANTE LA INFECCIÓN EXPERIMENTAL CON TRYPANOSOMA CRUZI. Characterization of the cardiac innate immune response during Trypanosoma cruzi experimental infection.

La enfermedad de Chagas, causada por Trypanosoma cruzi, constituye la principal miocarditis infecciosa a nivel mundial. Crecientes evidencias revelan que la respuesta inmune innata tendría un rol determinante en la fisiopatología de las enfermedades cardiovasculares. La inmunidad innata es la primera línea de defensa, no específica, preprogramada para combatir agentes infecciosos. Este sistema censa la presencia de antígenos extraños a través de los receptores tipo toll (TLR) produciendo citoquinas y activando mecanismos microbicidas. Sin embargo, los TLRs también se hayan distribuidos en las células parenquimales no inmunes, jugando un importante rol tanto en la defensa como en la homeostasis de cada tejido. Durante la etapa aguda de la infección, el T. cruzi invade y se replica dentro de una amplia variedad de células y tejidos. Pero posteriormente, los parásitos son efectivamente eliminados de la mayoría de los tejidos persistiendo durante toda la vida en las células del músculo cardíaco y esquelético de los pacientes infectados. Debido a que el mantenimiento de la célula cardíaca infectada es crítica para la patogénesis de la enfermedad, los mecanismos que participan en la sobrevida de los cardiomiocitos están siendo foco de nuestro estudio. Hemos demostrado, que la infección ejerce efectos antiapoptóticos sobre células cardíacas aisladas. Nuestra hipótesis es que la inmunidad innata cardíaca estaría involucrada en el mantenimiento de la sobrevida de los miocitos así como en la defensa contra el parásito. Objetivo general: determinar la participación de la respuesta inmune innata cardíaca en el desarrollo de la enfermedad de Chagas experimental murina. Objetivos específicos: 1) Analizar el compromiso de TLRs en la respuesta anti-apoptótica y de autofagia de cardiomiocitos aislados de ratones salvajes y de ratones deficientes en TLR4, TLR2 y en MyD88, molécula adaptadora de la señalización por TLRs, sometidos a la infección con el parásito. 2) Determinar la importancia de la actividad cisteín proteasa parasitaria en el grado de infectividad y la sobrevida de cultivos primarios de ratones salvajes infectados con parásitos transgénicos que poseen disminuída o nula actividad cisteín proteasa. 3) Establecer la cinética de expresión de TLR2/TLR6, TLR4 y TLR9, factores antiapoptóticos (Bcl-2, Bcl-xL, etc.), daño cardíaco y la carga parasitaria en el tejido cardíaco de ratones infectados salvajes y/o deficientes antes mencionados. Materiales y Métodos: Los animales serán infectados i.p. con 5x103 parásitos y se determinará la cinética de expresión de los mediadores mencionados por western blot e inmunofluorescencia, la carga parasitaria será determinada por qRT-PCR. Como controles se procesarán animales inyectados con solución salina. En cultivos primarios de cardiomiocitos de ratones neonatos salvajes y deficientes infectados se estudiará la carga parasitaria, la activación de los mecanismos microbicidas (producción de óxido nítrico, metabolitos reactivos del oxígeno y del nitrógeno, ciclooxigenasa, etc.), producción de citoquinas y expresión de moléculas anti-apoptóticas (Bcl-2, Bcl-xL, Bax, etc.). Se explorará la tasa de apoptosis en cultivos deprivados de suero. La autofagia se analizará por microscopia electrónica. Cultivos controles serán mantenidos en medio o tratados con ligandos de los diferentes TLRs. Resultados preliminares sugieren que tanto TLR2 como Bcl-2 se incrementan en tejido cardíaco infectado. Esto nos lleva a profundizar en los mecanismos observados en cultivos y estudiarlos en un modelo in vivo, analizando la posible importancia que tiene la inmunidad innata cardíaca en el control del establecimiento de la infección. La comprensión de los mecanismos que mantienen la sobrevida de los cardiomiocitos y su respuesta a la infección es importante ya que el conocimiento de las bases moleculares es fundamental para el desarrollo de nuevos agentes quimioterapéuticos. Chagas disease is endemic in Central and South America and causes the most common myocarditis worldwide. We have previously reported that the cardiotrophic parasite Trypanosoma cruzi, its etiological agent, protects cardiomyocytes against apoptosis induced by growth factor deprivation activating the PI3K/Akt and MEK1/ERK signaling pathways. Recent studies have shown that local innate immunity plays a key role in initiating and coordinating homeostatic as well as defense responses in the heart. One of the mechanisms by which the innate immune system senses the presence of foreign antigens is through TLRs. The stimulation of these receptors leads to the activation and nuclear translocation of NF-kB transcription factor and the production of cytokines. Proinflammatory cytokines, in turn, appear to play a central role in the orchestration and timing of the intrinsic cardiac stress response providing, under different situations, instantaneous anti-apoptotic cytoprotective signals, which allow tissue repair and/or remodeling. The aim of the present project is to study the cardiomyocyte innate immune responses to T. cruzi infection and its role in target cell protection from apoptosis. Specific objectives: 1) Study the mechanism triggered by TLR in the anti-apoptotic response and parasite load of infected cardiomyocyte primary cultures from wild type and mice deficient in TLR2, TLR4 or MyD88. 2) Determine the effect of parasite cisteín protease activity on primary cultures from wild type mice. 3) Determine the TLR signaling-involvement in parasite load and survival indicators in deficient mice. Preliminary results showed us that cardiac-TLR2 may be involved in the anti-apoptotic effect elicited by the parasite and prompted us to establish the mechanisms triggered by the innate immunity that mediate parasite persistence within the host cell.

A simple method to purify biologically and antigenically preserved bloodstream trypomastigotes of Trypanosoma cruzi using Deae-cellulose columns

A method to purify trypanosomastigotes of some strains of Trypanosoma cruzi (Y, CL, FL, F, "Berenice", "Colombiana" and "São Felipe") from mouse blood by using DEAE-cellulose columns was standardized. This procedure is a modification of the Lanham & Godfrey methods and differs in some aspects from others described to purify T. cruzi bloodstream trypomastigotes, mainly by avoidance of prior purifications of parasites. By this method, the broad trypomastigotes were mainly isolated, accounting for higher recoveries obtained with strains having higher percentages of these forms: processing of infected blood from irradiated mice could be advantageous by increasing the recovery of parasites (percentage and/or total number) and elution of more slender trypomastigotes. Trypomastigotes purified by this method presented normal morphology and motility, remained infective to triatomine bugs and mice, showing in the latter prepatent periods and courses parasitemia similar to those of control parasites, and also reproducing the polymorphism pattern of each strain. Their virulence and pathogenicity also remained considerably preserved, the latter property being evaluated by LD 50 tests, mortality rates and mean survival time of inoculated mice. Moreover, these parasites presented positive, clear and peripheral immunofluorescence reaction at titres similar to those of control organisms, thus suggesting important preservation of their surface antigens.

Evaluation of a monoclonal antibody affinity purified antigen for zymodeme specific serological diagnosis of Trypanosoma cruzi infection

Theoretically, serological assays with affinity purified marker antigens can allow strain-specific diagnosis even when parasites cannot be retrieved from and infected host. A Trypanosoma cruzi antigen was purified by affinity chromatography using a zymodeme (Z) 2 specific monoclonal antibody (2E2C11). An indirect enzyme-linked immunosorbent assay (ELISA) based on the purified antigen could discriminate between sera from rabbits immunized with T. cruzi zymodeme clones but could not discriminate between sera from mice infected with different zymodemes.

An Investigation on the Ecology of Triatoma vitticeps (Stal, 1859) and its Possible Role in the Transmission of Trypanosoma cruzi, in the Locality of Triunfo, Santa Maria Madalena Municipal District, State of Rio de Janeiro, Brazil

From January 1989 to April 1995, 465 specimens of Triatoma vitticeps were collected in the locality of Triunfo, 2nd District of Santa Maria Madalena municipal district, State of Rio de Janeiro. The bugs were found indoors by local residents with predominance of adults. The flight activity was high in hot months when the incidence in the domicile also increased. Two hundred and two bugs (111 alive and 91 dead) were examined for Trypanosoma cruzi infection. This was detected in 31 of the dead bugs (34%) and 88 (79%) of the live bugs examined. With a view to investigate the possible vertebrate hosts of the T. cruzi isolates, the blood of 122 mammals was examined through Giemsa-stained smears, hemocultures and xenodiagnosis. T. cruzi was detected in three specimens of Didelphis marsupialis and T. (M.) theileri was detected in one specimen of Bos taurus. The parasites were isolated from triatomine feces, xenoculture and hemoculture. No evidence of human infection was detected in 58 inhabitants examined, as evaluated by indirect imunofluorescence technique using T. cruzi epimastigotes as antigens. These results show that T. vitticeps is still a sylvatic species although nymphs have been found inside the domicile. Thus, an epidemiological vigilance is necessary to know the behaviour of this species following the continuous modifications promoted by the presence of man.

Trypanosoma cruzi: Maintenance in Culture Modify Gene and Antigenic Expression of Metacyclic Trypomastigotes

In this study we examined whether the maintenance of Trypanosoma cruzi by long-time in axenic culture produces changes in gene expression and antigenic profiles. The studies were made with a Dm30L-clone from a low-virulent strain and a non-cloned virulent EP-strain of T. cruzi. Both parasites were maintained, for at least seven years, by successive alternate passage triatomine/mouse (triatomine condition), or by serial passage in axenic medium (culture condition). The comparison of the [35S]methionine metabolic labeling products of virulent and non-virulent parasites by 2D-SDS-PAGE, clearly indicates that the expression of metacyclic trypomastigotes (but not of epimastigotes) proteins have been altered by laboratory maintenance conditions. Western blot analysis of EP and Dm30L-epimastigotes using a serum anti-epimastigotes revealed that although most of antigens are conserved, four antigens are characteristics of triatomine condition parasites and three other are characteristics of culture condition parasites. Anti-metacyclics serum revealed significative differences in EP- and Dm30L-metacyclic trypomastigotes from triatomine condition. However, avirulent metacyclic forms were antigenically very similar. These results suggest that besides a possible selection of avirulent subpopulation from T. cruzi strains genetically heterogeneous when maintained by long time in axenic culture, changes in virulence might be due to post-translational modifications of the antigens induced by the absence of the natural alternability (vertebrate-invertebrate) in the life-cycle of T. cruzi

Humoral Immune Response Kinetics in Philander opossum and Didelphis marsupialis Infected and Immunized by Trypanosoma cruzi Employing an Immunofluorescence Antibody Test

Philander opossum and Didelphis marsupialis considered the most ancient mammals and an evolutionary success, maintain parasitism by Trypanosoma cruzi without developing any apparent disease or important tissue lesion. In order to elucidate this well-balanced interaction, we decided to compare the humoral immune response kinetics of the two didelphids naturally and experimentally infected with T. cruzi and immunized by different schedules of parasite antigens, employing an indirect fluorescence antibody test (IFAT). Both didelphids responded with high serological titers to different immunization routes, while the earliest response occurred with the intradermic route. Serological titers of naturally infected P. opossum showed a significant individual variation, while those of D. marsupialis remained stable during the entire follow-up period. The serological titers of the experimentally infected animals varied according to the inoculated strain. Our data suggest that (1) IFAT was sensitive for follow-up of P. opossum in natural and experimental T. cruzi infections; (2) both P. opossum and D. marsupialis are able to mount an efficient humoral immune response as compared to placental mammals; (3) experimentally infected P. opossum and D. marsupialis present distinct patterns of infection, depending on the subpopulation of T. cruzi, (4) the differences observed in the humoral immune responses between P. opossum and D. marsupialis, probably, reflect distinct strategies selected by these animals during their coevolution with T. cruzi.

Integration of Trypanosoma cruzi kDNA minicircle sequence in the host genome may be associated with autoimmune serum factors in Chagas disease patients

Integration of kDNA sequences within the genome of the host cell shown by PCR amplification with primers to the conserved Trypanosoma cruzi kDNA minicircle sequence was confirmed by Southern hybridization with specific probes. The cells containing the integrated kDNA sequences were then perpetuated as transfected macrophage subclonal lines. The kDNA transfected macrophages expressed membrane antigens that were recognized by antibodies in a panel of sera from ten patients with chronic Chagas disease. These antigens barely expressed in the membrane of uninfected, control macrophage clonal lines were recognized neither by factors in the control, non-chagasic subjects nor in the chagasic sera. This finding suggests the presence of an autoimmune antibody in the chagasic sera that recognizes auto-antigens in the membrane of T. cruzi kDNA transfected macrophage subclonal lines.

Human chronic chagasic cardiopathy: participation of parasite antigens, subsets of lymphocytes, cytokines and microvascular abnormalities

This article tries to demonstrate by new pathological findings (with the use of immunohistochemical technique and confocal laser microscopy) that chronic chagasic cardiomyopathy is a result of multiple factors involving myocarditis, immunodepression, severe fibrosis and microvessels dilatation and that all of these alterations are probably directly related with the presence of Trypanosoma cruzi parasites in the host associated with inadequate immunological response of the host.

Integrate Study of a Bolivian Population Infected by Trypanosoma cruzi, the Agent of Chagas Disease

A cross section of a human population (501 individuals) selected at random, and living in a Bolivian community, highly endemic for Chagas disease, was investigated combining together clinical, parasitological and molecular approaches. Conventional serology and polymerase chain reaction (PCR) indicated an active transmission of the infection, a high seroprevalence (43.3%) ranging from around 12% in < 5 years to 94.7% in > 45 years, and a high sensitivity (83.8%) and specificity of PCR. Abnormal ECG tracing was predominant in chagasic patients and was already present among individuals younger than 13 years. SAPA (shed acute phase antigen) recombinant protein and the synthetic peptide R-13 were used as antigens in ELISA tests. The reactivity of SAPA was strongly associated to Trypanosoma cruzi infection and independent of the age of the patients but was not suitable neither for universal serodiagnosis nor for discrimination of specific phases of Chagas infection. Anti-R-13 response was observed in 27.5% only in chagasic patients. Moreover, anti-R13 reactivity was associated with early infection and not to cardiac pathology. This result questioned previous studies, which considered the anti-R-13 response as a marker of chronic Chagas heart disease. The major clonets 20 and 39 (belonging to Trypanosoma cruzi I and T. cruzi II respectively) which circulate in equal proportions in vectors of the studied area, were identified in patients' blood by PCR. Clonet 39 was selected over clonet 20 in the circulation whatever the age of the patient. The only factor related to strain detected in patients' blood, was the anti-R-13 reactivity: 37% of the patients infected by clonet 39 (94 cases) had anti-R13 antibodies contrasting with only 6% of the patients without clonet 39 (16 cases).

High prevalence anti-Trypanosoma cruzi antibodies, among blood donors in the State of Puebla, a non-endemic area of Mexico

Blood transfusion is the second most common transmission route of Chagas disease in many Latin American countries. In Mexico, the prevalence of Chagas disease and impact of transfusion of Trypanosoma cruzi-contaminated blood is not clear. We determined the seropositivity to T. cruzi in a representative random sample, of 2,140 blood donors (1,423 men and 647 women, aged 19-65 years), from a non-endemic state of almost 5 millions of inhabitants by the indirect hemagglutination (IHA) and enzyme linked immunosorbent assay (ELISA) tests using one autochthonous antigen from T. cruzi parasites, which were genetically characterized like TBAR/ME/1997/RyC-V1 (T. cruzi I) isolated from a Triatoma barberi specimen collected in the same locality. The seropositivity was up to 8.5% and 9% with IHA and ELISA tests, respectively, and up to 7.7% using both tests in common. We found high seroprevalence in a non-endemic area of Mexico, comparable to endemic countries where the disease occurs, e.g. Brazil (0.7%), Bolivia (13.7%) and Argentina (3.5%). The highest values observed in samples from urban areas, associated to continuous rural emigration and the absence of control in blood donors, suggest unsuspected high risk of transmission of T. cruzi, higher than those reported for infections by blood e.g. hepatitis (0.1%) and AIDS (0.1%) in the same region.

Antibody isotype responses in Balb/c mice immunized with the cytoplasmic repetitive antigen and flagellar repetitive antigen of Trypanosoma cruzi

In the present report we analyzed the levels of IgG1, IgG2a, IgG2b and IgG3 isotypes from Balb/c mice immunized with cytoplasmic repetitive antigen (CRA), and flagelar repetitive antigen (FRA) of Trypanosoma cruzi. The immunization was done by subcutaneous route three times (20 days apart) and the analysis was performed 14 days after each treatment. CRA-immunized mice produced high levels of all IgG isotypes, mainly IgG3 and IgG1. FRA-immunization elicited only high levels of IgG1.

Analysis of expressed sequence tags from Trypanosoma cruzi amastigotes

A total of 880 expressed sequence tags (EST) originated from clones randomly selected from a Trypanosoma cruzi amastigote cDNA library have been analyzed. Of these, 40% (355 ESTs) have been identified by similarity to sequences in public databases and classified according to functional categorization of their putative products. About 11% of the mRNAs expressed in amastigotes are related to the translational machinery, and a large number of them (9% of the total number of clones in the library) encode ribosomal proteins. A comparative analysis with a previous study, where clones from the same library were selected using sera from patients with Chagas disease, revealed that ribosomal proteins also represent the largest class of antigen coding genes expressed in amastigotes (54% of all immunoselected clones). However, although more than thirty classes of ribosomal proteins were identified by EST analysis, the results of the immunoscreening indicated that only a particular subset of them contains major antigenic determinants recognized by antibodies from Chagas disease patients.