Total Leishmania antigens with Poly(I:C) induce Th1 protective response.

Our proposal was to develop a vaccine based on total Leishmania antigens (TLA) adjuvanted with polyinosinic-polycytidylic acid [Poly(I:C)] able to induce a Th1 response which can provide protection against Leishmania infection. Mice were vaccinated with two doses of TLA-Poly(I:C) administered by subcutaneous route at 3-week interval. Humoral and cellular immune responses induced by the immunization were measured. The protective efficacy of the vaccine was evaluated by challenging mice with infective promastigotes of Leishmania (Leishmania) amazonensis into the footpad. Mice vaccinated with TLA-Poly(I:C) showed a high anti-Leishmania IgG titre, as well as increased IgG1 and IgG2a subclass titres compared with mice vaccinated with the TLA alone. The high IgG2a indicated a Th1 bias response induced by the TLA-Poly(I:C) immunization. Accordingly, the cellular immune response elicited by the formulation was characterized by an increased production of IFN-γ and no significant production of IL-4. The TLA-Poly(I:C) immunization elicited good protection, which was associated with decreased footpad swelling, a lower parasite load and a reduced histopathological alteration in the footpad. Our findings demonstrate a promising vaccine against cutaneous leishmaniasis that is relatively economic and easy to develop and which should be taken into account for preventing leishmaniasis in developing countries.

In vitro activity of N-benzenesulfonylbenzotriazole on Trypanosoma cruzi epimastigote and trypomastigote forms.

Chagas disease is still an important health problem in Central and South America. However, the only drugs currently available for specific treatment of this disease may induce toxic side effects in the host. The aim of this work was to determine the activity of N-benzenesulfonylbenzotriazole (BSBZT) against the protozoan parasite Trypanosoma cruzi. The effects of BSBZT and benzotriazole (BZT) were compared to those of benznidazole (BZL) on epimastigote and trypomastigote forms. BSBZT was found to have an in vitro growth inhibitory dose-dependent activity against epimastigotes, with flow cytometry analysis confirming that the treated parasites presented size reduction. BSBZT showed an IC(50) of 21.56 µg/mL (81.07 µM) against epimastigotes at 72 h of incubation, whereas BZT did not affect the growth of this parasite form. Furthermore, the toxic effect of BSBZT, was stronger and appeared earlier (at 24h) in trypomastigotes than in epimastigotes, with the LC(50) of this compound being 28.40 µg/mL (106.79 µM) against trypomastigotes. The concentrations of BSBZT used in this study presented low hemolytic activity and cytotoxicity. Consequently, at concentrations near IC(50) and LC(50) (25µg/mL), BSBZT caused only 2.4% hemolysis and 15% of RAW 264.7 cell cytotoxicity. These results reveal the potential of BSBZT as a prototype in drug design for developing new anti-T. cruzi compounds.

Burnout and engagement among resident doctors in the Netherlands: a national study.

OBJECTIVES: This study was performed in order to gather insight into the well-being of Dutch medical residents. METHODS: In 2005, all Dutch residents registered through the Medical Registration Committee (n = 5245) were sent a self-report questionnaire to assess socio-demographic and work-related characteristics, burnout and engagement. RESULTS: Of the 5140 eligible residents, 2115 completed the questionnaire (41%). Of those, 21% fulfilled the criteria for moderate to severe burnout and 27% were highly engaged with their work. Women reported more emotional exhaustion and less depersonalisation than men; age was weakly but significantly related to depersonalisation, and married residents and parents reported less depersonalisation than their single or childless counterparts. More men than women were found to be highly engaged and men specifically reported more vigour. Number of years in training was weakly but significantly related to absorption. With regard to occupational risk factors, significant between-group differences were found for the effects of clinical setting on emotional exhaustion, engagement, vigour and absorption. Residents in training in a mental health clinic were most emotionally exhausted and those in a rehabilitation centre were least engaged. General surgery represented the specialty with the lowest number of residents suffering from burnout, followed by obstetrics and gynaecology and any supportive specialty. General surgery residents were also found to be more highly engaged, vigorous, dedicated and absorbed than others. CONCLUSIONS: As more than a fifth of the medical residents who responded could be diagnosed as suffering from burnout, we conclude that this problem needs addressing in the Dutch health care system, especially given that a relationship was proven between burnout and suboptimal patient care. We must look for solutions and interventions which will improve the work situation of medical residents. Striving for healthy workers in health care has to become daily practice.

The relationship between reciprocity and burnout in Dutch medical residents.

OBJECTIVE: This study examined reciprocity in medical residents' relationships with supervisors, fellow residents, nurses and patients, and associations between reciprocity and burnout. Furthermore, we considered if a discrepancy between the perceived and preferred levels of reciprocity influenced the level of burnout complaints. METHODS: In 2003, self-report questionnaires were sent to the homes of all 292 medical residents at the University Medical Centre Groningen (UMCG), Groningen, the Netherlands. Reciprocity was measured with a single-item reciprocity scale based on the Hatfield Global Measure of Equity Scale. The Utrecht Burn-Out Scale (UBOS/MBI-HHS) was used to measure burnout. RESULTS: A total of 158 residents participated in the study. Those who reported under-benefiting in the relationship with supervisors perceived significantly more emotional exhaustion and depersonalisation than those who perceived a reciprocal relationship. Residents who indicated that they over-benefited in the relationship with nurses reported more emotional exhaustion than residents who perceived a reciprocal relationship and less personal accomplishment than residents who perceived a reciprocal relationship or under-benefit. No differences on the burnout subscales were found between residents who perceived their relationships with patients and fellow residents to be reciprocal and those who considered they under- or over-benefited. The greater the discrepancy between perceived and preferred reciprocity in the relationship with the supervisor, the more emotional exhaustion residents reported. CONCLUSIONS: Perceptions of reciprocity in relationships with supervisors and nurses had particular influence on the level of burnout complaints among residents. The discrepancy between the impacts of perceived and preferred reciprocity on burnout was negligible and the only significant relationship to emerge concerned that with emotional exhaustion.

Cruzipaína, la principal cisteín proteasa de trypanosoma cruzi en la interrelación huésped-parásito / Cruzipain, a major Trypanosoma cruzi cystein protease in the host-parasite interplay

La enfermedad de Chagas, causada por el parásito protozoarioTrypanosoma cruzi, es endémica en América Central y Sudaméricay representa la miocarditis más frecuente a nivel mundial.El establecimiento de la infección crónica conduce a una patologíacardíaca debilitante por la cual mueren más de 50,000 personascada año. No existe consenso sobre si la causa del daño tisulares ocasionada por el parásito o está exacerbada por una respuestaautoinmune. En ambos escenarios, ha sido sugerido quecruzipaína- la principal cisteín proteasa del T. cruzi- cumple unrol importante en la progresión de la enfermedad.Cruzipaína, miembro de la superfamilia de las papaínas, seexpresa como una mezcla compleja de isoformas en los diferentesestadíos de desarrollo de todas las cepas del parásito. Esta glicoproteínaparticipa en la internalización del T. cruzi en las célulasmamíferas, lo que ha sido demostrado con inhibidores específicosde la enzima que interfieren en la invasión celular y la replicacióndel parásito. Además, cruzipaína genera una fuerte respuestaimmune en individuos infectados. Estas característicashacen de cruzipaína un potencial blanco de drogas terapéuticas.La presente revisión resume el conocimiento actual sobre elrol de cruzipaína en la patogénesis de la enfermedad, su compromisoen la invasión de células del huésped así como su participaciónen la activación y evasión de la respuesta inmune enmodelos experimentales y en pacientes chagásicos. El avance enesta área de investigación, proveerá nuevas estrategias terapéuticastendientes a incrementar la respuesta inmunoprotectiva yprevenir la respuesta deletérea producida por el parásito

The protozoan parasite Trypanosoma cruzi, etiological agentof Chagas disease, is endemic in Central and South America andproduces the most common myocarditis worldwide.Parasite persistence eventually leads to a debilitating heartdisease that kills more than 50,000 people every year. There is noconsensus as to whether tissue damage is caused entirely by theparasite or is exacerbated by an autoimmune response. In bothmodels of disease progression, cruzipain- the major cysteine proteinaseof T. cruzi- has been suggested to play an important role.Cruzipain is a member of the papain superfamily, and it isexpressed as a complex mixture of isoforms by different strains ofthe parasite, as well as in all its developmental stages. This parasiteglycoprotein plays a role in the process of T. cruzi internalizationinto mammalian cells, as proved by specific enzyme inhibitors, whichinterfere with cell invasion and inhibit parasite replication.In addition, cruzipain not only is essential for parasite survivalbut also generates a strong immune response in infected individuals.These characteristics point to cruzipain as a potential targetfor drug therapy and for the generation of immune responses.This review analyses our present knowledge of the role ofcruzipain in the disease pathogenesis, its involvement in host cellinvasion, immune activation and evasion by T. cruzi in experimentalmodels and human infection. Ongoing studies in this researcharea may provide novel therapeutic strategies that couldenhance the immunoprotective response while preventing thedeleterious parasite elicited responses observed during Chagasdisease

Trypanosoma cruzi: chemotherapeutic effects of clomipramine in mice infected with an isolate obtained from an endemic area.

The susceptibility of Trypanosoma cruzi strains to nifurtimox and benznidazole has been investigated and resistant strains have been described. Some tricyclic drugs are lethal for trypomastigote and epimastigote forms of T. cruzi (Tulahuen strain) and prevent the disease in mice. We investigated whether clomipramine, a tricyclic antidepressant drug with anti-trypanothione reductase and anti-calmodulin effects, could be effective in treating Albino Swiss mice infected with trypomastigotes of a new T. cruzi isolate from a chronic patient from an endemic area of Argentina in two different treatment schedules. Both treatment schedules were effective in reducing electrocardiographic changes and preventing myocardial structural damage. The cardiac beta-receptors low affinity was compensated for by an increment in their density. This probably maintained cardiac function since 70% of the mice survived for more than 2 years even though anti-cruzipain titers remained high. These results demonstrate that clomipramine, clinically used as a neuroleptic, could be a promising trypanocidal agent for the treatment of Chagas' disease.

Thioridazine treatment prevents cardiopathy in Trypanosoma cruzi infected mice.

Trypanosoma cruzi trypanothione reductase is an enzyme that has been identified as a potential target for chemotherapy. Thioridazine inhibits it and prevented cardiopathy in mice infected with T. cruzi Tulahuen strain. As not all T. cruzi strains respond to treatment in the same way, an isolate from a chronic patient (SGO Z12) was used; parasitaemias were studied along with, survival, serology, electrocardiography, histology and cardiac beta receptor function. Parasitaemia in thioridazine (80 mg/(kg day) for 3 days) treated mice was less and lasted for a shorter period (P < 0.01), there were reduced electrocardiographic and histological alterations and significantly improved survival (80% of non-treated died). Treated mice had lower receptor affinity and higher density as a compensatory mechanism, modifying the course of T. cruzi infection (SGO Z12 isolate) and preventing the consequent cardiopathy.

Antibodies to an epitope from the Cha human autoantigen are markers of Chagas' disease.

Chagas' disease is a prevalent disease in South America that is thought to have an autoimmune etiology. We previously identified human Cha as a new autoantigen recognized by chagasic sera. Those sera recognized an epitope spanning amino acids 120 to 129 of Cha, named R3. In the present study we have used the synthetic R3 peptide for the detection of serum immunoglobulin G antibodies from patients at different stages of Chagas' disease, including a therapeutically treated group. The immunoreactivity with R3 by enzyme-linked immunosorbent assay (ELISA) showed 92.4% sensitivity and 100% specificity for Chagas' disease sera. This sensitivity and specificity were higher than for any other autoantigen described to date. No anti-R3 antibodies were detected in sera from Leishmania-infected or idiopathic dilated cardiomyopathy patients or healthy controls from the same areas. Moreover, anti-R3 antibody reactivity detected by ELISA correlated with conventional serological tests as indirect immunofluorescence and ELISA assays with Trypanosoma cruzi extracts and other diagnostic tests as indirect hemagglutination. The levels of anti-R3 antibodies increased with progression and symptomatology of Chagas' disease. More interestingly, a statistically significant fall in anti-R3 antibody titer was observed in patients treated with antiparasitic drugs. Those results suggest that the presence of anti-R3 antibodies is a highly specific marker of Chagas' disease and that R3 ELISA could be helpful in the diagnosis and monitoring of this disease.

Effects of clomipramine on Trypanosoma cruzi infection in mice.

Trypanosoma cruzi, widely distributed in Latin American countries, provokes Chagas disease, characterized by cardiomyopathy and mega-viscera. The drugs used currently for treatment of acute Chagas disease are highly toxic; the side-effects are undesirable and patients may abandon treatment. We have previously demonstrated that clomipramine (CLO) exerts trypanocidal effects upon epimastigotes and trypomastigotes in vitro with anticalmodulin activity. The present study analyses the effectiveness of CLO treatment in mice infected with a low number of T. cruzi, an animal model that reproduces acute, indeterminate and chronic phases of this trypanosomiasis. In this work, our results demonstrated that CLO 5 mg/kg daily for 30 days, or 2 doses of CLO 40 mg/kg given intraperitoneally at 1 h and 7 days after infection, was not toxic for the host, but was effective against the parasite in that parasitaemias became negative and only mild heart structural and electrocardiographic alterations were detected in the chronic phase in the group treated with CLO 5 mg/kg. In mice treated with CLO 40 mg/kg, none of these alterations was detected. Cardiac beta receptor density and affinity returned to normal in the chronic stage in both experimental groups. T. cruzi enzymes such as calmodulin and trypanothione reductase represent potential drug targets. It has been reported that both can be inhibited by CLO, a tricyclic drug used in clinical therapeutics. We have shown that CLO strongly decreased the mortality rate and electrocardiographic alterations; in addition cardiac beta receptor density and heart histology returned to, or close to, normality 135 days post infection. These results clearly demonstrated that CLO treatment modified significantly the natural evolution of T. cruzi infection.

Anti-galectin-1 autoantibodies in human Trypanosoma cruzi infection: differential expression of this beta-galactoside-binding protein in cardiac Chagas' disease.

The pathogenesis of Chagas' disease has been subject of active research and still remains to be ascertained. Galectin-1 (Gal-1), a member of a conserved family of animal beta-galactoside-binding proteins, localized in human heart tissue, has been suggested to play key roles in immunological and inflammatory processes. In the present study we demonstrated the occurrence of anti-Gal-1 autoAb in sera from patients in the acute and chronic stages of Chagas' disease (ACD and CCD) by means of ELISA and Western blot analysis. We found a marked increase in the level and frequency of Ig E anti-Gal-1 antibodies in sera from patients with ACD, but a low frequency of Ig M anti-Gal-1 immunoreactivity. Moreover, Ig G immunoreactivity to this beta-galactoside-binding protein was found to be correlated with the severity of cardiac damage in CCD, but was absent in nonrelated cardiomyopathies. We could not detect immunoreactivity with Trypanosoma cruzi antigens using a polyclonal antibody raised to human Gal-1 and no hemagglutinating activity could be specifically eluted from a lactosyl-agarose matrix from parasite lysates. Moreover, despite sequence homology between Gal-1 and shed acute phase antigen (SAPA) of T. cruzi, anti-Gal-1 antibodies eluted from human sera failed to cross-react with SAPA. In an attempt to explore whether Gal-1 immunoreactivity was originated from endogenous human Gal-1, we finally investigated its expression levels in cardiac tissue (the main target of Chagas' disease). This protein was found to be markedly upregulated in cardiac tissue from patients with severe CCD, compared to cardiac tissue from normal individuals.

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.

Cruzipain induces autoimmune response against skeletal muscle and tissue damage in mice.

The goal of the current study was to investigate whether cruzipain, a major Trypanosoma cruzi antigen, is able to induce in mice an autoimmune response and skeletal muscle damage. We demonstrate that immunization with cruzipain triggers immunoglobulin G antibody binding to a 210-kDa antigen from a syngeneic skeletal muscle extract. The absorption of immune sera with purified myosin completely eliminated this reactivity, confirming that the protein identified is really myosin. We also found that spleen cells from immunized mice proliferated in response to a skeletal muscle extract rich in myosin and to purified myosin. Cells from control mice did not proliferate against any of the antigens tested. In addition, we observed an increase in plasma creatine kinase activity, a biochemical marker of muscle damage. Histological studies showed inflammatory infiltrates and myopathic changes in skeletal muscle of immunized animals. Electromyographic studies of these mice revealed changes such as are found in inflammatory or necrotic myopathy. Altogether, our results suggest that this experimental model provides strong evidence for a pathogenic role of anticruzipain immune response in the development of muscle tissue damage.

Inflammatory arthritic process, iridocyclitis and immune response to articular and ocular antigens in Wistar rats injected with T. gondii trophozoites.

The present study deals with the potential role of T. gondii in inducing an arthritic inflammatory process. Wistar rats were injected subcutaneously (sc) into the right footpad with viable T. gondii trophozoites emulsified in incomplete Freund's adjuvant (IFA). The control group was injected with IFA. All parasite-injected animals developed a local inflammatory process characterized by hind limb swelling and marked restriction of ankle motility approximately 25 days after injection. Histopathogical studies of the joints, carried out 90 days after injection, revealed intense mononuclear infiltration, proliferation of granulation tissue, giant cells and necrosis in the synovia of 90% of T. gondii-injected rats. Strikingly, 40% (4/10) of the parasite-injected animals developed iridocyclitis, which was characterized by intense mononuclear infiltration around the iris-ciliary microvasculature in two animals and a slightly pronounced infiltrate of polymorphonuclear and mononuclear cells in two other animals. Antibodies to soluble T. gondii antigens (STAg) were detected in all parasite-injected rats. Antibodies against articular and ocular antigens such as proteoglycans, type II collagen, retinal S antigen and iris antigens were detected by ELISA in 40, 80, 70 and 70% of T. gondii -injected animals, respectively. Control animals injected with IFA failed to develop any articular or ocular process or humoral immune response. The present study demonstrated that footpad sc injection of Wistar rats with viable T. gondii trophozoites was able to induce a localized inflammatory arthritic process which, in some of the animals, was accompanied by iridocyclitis and immune response against articular and ocular components.

Thioridazine treatment modifies the evolution of Trypanosoma cruzi infection in mice.

Thioridazine, a tricyclic drug, is known to have a direct effect on Trypanosoma cruzi, disrupting the parasites' mitochondria and kinetoplasts. In the present study, the drug was used orally, at 80 mg/kg.day for 3 days, to treat mice inoculated with low numbers of T. cruzi. The drug caused no apparent toxicity in the host. It cleared trypomastigotes from the bloodstream, prevented the histological and functional alterations of the heart normally observed in the chronic phase of the experimental disease, and greatly reduced the mortality rate compared with that in untreated, infected controls. When checked 135 days post-infection, the density of cardiac beta receptors and the cardiac histology of the treated mice were indistinguishable from those of uninfected, untreated controls. The drug is already used to treat humans, as a neuroleptic drug. It appears to be able to prevent acute infection with T. cruzi evolving into chronic disease, at least in mice, and may be a useful base from which to design new agents for the treatment of Chagas disease.

Trypanosoma cruzi: the major cysteinyl proteinase (cruzipain) is a relevant immunogen of parasite acidic antigens (FIII).

This study examined the immune responses induced by cruzipain, a well-characterized T. cruzi antigen, to determine whether it is a relevant immunogen among the parasite acidic antigens (FIII), for which some biological properties have been studied previously. Humoral and cellular immune responses were investigated in BALB/C mice after immunization with cruzipain or FIII. Skin tests revealed immediate type-hypersensitivity (ITH) and delayed-type hypersensitivity (DTH) reactions to cruzipain in both groups of immunized mice. IgG1 and IgE isotypes against cruzipain were detected by ELISA in both groups and immunoblot studies showed that these antibodies recognized a major protein band of 50 kDa, cruzipain. The antigen-specific proliferative responses of spleen lymphocytes from both groups of immunized mice were also increased. Immunization with cruzipain of FIII antigen significantly enhanced the percentage survival of mice challenged with 10(3) trypomastigotes. The results revealed high cross-reactivity between cruzipain and FIII, suggesting the cruzipain is a relevant immunogen among the parasite acidic antigens.

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.

Trypanosoma cruzi: immunological cross-reactivity of the major cysteinyl proteinase (cruzipain) with a parasite cytosol acidic antigen (FIV).

This paper shows that a polyclonal monospecific rabbit antiserum to cruzipain, the major T. cruzi cystein proteinase, cross-reacts with a cytosol acidic antigen (F IV) isolated from the epimastigote stage of the same parasite. In addition, antibodies specific for F IV purified from chagasic patient sera or murine anti F IV sera, also react with cruzipain. This was demonstrated by ELISA, DOT-ELISA, native and electrophoretic Immunoblot. These findings suggest that F IV contains an antigen immunologically cross-reactive with cruzipain.

Antibody isotypes profiles against Trypanosoma cruzi acidic antigens in two Amerindian populations from a Chagas' disease endemic area.

The isotype distribution of the antibody response against one Trypanosoma cruzi antigenic fraction, FIV, and the putative association to heart disease were analyzed in patients of two apparently genetically distinct Amerindian populations, Mataco (M) and Toba (T), infected with this parasite. The isotypes profiles were analyzed by ELISA, and the antigen specificity of IgG immune response was determined by the immunoblot method. The percentages of infected individuals with abnormal electrocardiograms (GII) were 50% for population M and 10% for population T. Many individuals from both populations had measureable IgG2, IgG3 and IgG4 antibodies to FIV, but the level and frequency (%) of positive sera in population T was considerably higher than in population M (70% vs 15% for IgG2; 75% vs 40% for IgG3; 85% vs 20% for IgG4). The level and frequency of IgG1 reactivity against FIV were similar in the two populations. When the sera were titrated, the most remarkable difference in isotype levels between populations T and M was seen for IgG2 and IgG4, the T population showing the highest titer. No association between clinical state and a particular isotype profile was found by ELISA in any population. When the antigen specificity of antibody response was determined by immunoblot, the antigen patterns recognized by sera from the two clinical groups showed some differences only in population M. All sera assayed from GII of population M fixed more IgG than those with normal electrocardiograms (GI). Two bands of 36 and 43 kD were revealed only in GII of this population. Similar antigenic patterns between the two clinical groups from population T were observed, and they were comparable with those obtained with GI from population M.

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' disease: IgG isotypes against Trypanosoma cruzi cytosol acidic antigens in patients with different degrees of heart damage.

We studied the reactivity of IgG isotypes detected in sera from chronic Chagas' disease patients with a Trypanosoma cruzi cytosol acidic antigenic fraction (F IV) and parasite epimastigote forms (EPI). All patients studied had positive serology for Chagas' disease, with normal electrocardiogram (Group I), abnormal ECG without cardiomegaly (Group II), and abnormal ECG with cardiomegaly (Group III). The highest levels of antibodies were observed in sera from Group II patients. A high prevalence of IgG1 and IgG3, low levels of IgG2, and IgG4 isotypes against EPI were found in sera from all groups by ELISA. When the F IV was used as antigen, IgG1 was the main antibody isotype detected by ELISA in all groups of patients. The antigenic recognition patterns by IgG1 among the different clinical groups by immunoblotting of F IV revealed some differences. The sera from Group I recognized antigens of F IV of 80, 53, and 43 kDa. Sera from Group III recognized mainly one antigenic band of 43 kDa. Finally, sera from Group II showed greater diversity of binding by IgG1, detecting between one and six bands in the 80 and 30 kDa ranges.