Co-infection with distinct Trypanosoma cruzi strains induces an activated immune response in human monocytes.

AIMS: The aim of the study was to evaluate the immune response triggered by the first contact of human monocytes with two T cruzi strains from distinct discrete typing units (DTUs) IV and V, and whether co-infection with these strains leads to changes in monocyte immune profiles, which could in turn influence the subsequent infection outcome. METHODS AND RESULTS: We evaluated the influence of in vitro single- and co-infection with AM64 and 3253 strains on immunological characteristics of human monocytes. Single infection of monocytes with AM64 or 3253 induced opposing anti-inflammatory and inflammatory responses, respectively. Co-infection was observed in over 50% of monocytes after 15 hours of culture, but this percentage dropped ten-fold after 72 hours. Co-infection led to high monocyte activation and an increased percentage of both IL-10 and TNF. The decreased percentage of co-infected cells observed after 72 hours was associated with a decreased frequency of TNF-expressing cells. CONCLUSION: Our results show that the exacerbated response observed in co-infection with immune-polarizing strains is associated with a decreased frequency of co-infected cells, suggesting that the activated response favours parasite control. These findings may have implications for designing new Chagas disease preventive strategies.

Development of chronic cardiomyopathy in canine Chagas disease correlates with high IFN-gamma, TNF-alpha, and low IL-10 production during the acute infection phase.

When infected with Trypanosoma cruzi, Beagle dogs develop symptoms similar to those of Chagas disease in human beings, and could be an important experimental model for a better understanding of the immunopathogenic mechanisms involved in chronic chagasic infection. This study evaluates IL-10, IFN-gamma and TNF-alpha production in the sera, culture supernatant, heart and cervical lymph nodes and their correlation with cardiomegaly, cardiac inflammation and fibrosis in Beagle dogs infected with T. cruzi. Pathological analysis showed severe splenomegaly, lymphadenopathy and myocarditis in all infected dogs during the acute phase of the disease, with cardiomegaly, inflammation and fibrosis observed in 83% of the animals infected by T. cruzi during the chronic phase. The data indicate that infected animals producing IL-10 in the heart during the chronic phase and showing high IL-10 production in the culture supernatant and serum during the acute phase had lower cardiac alterations (myocarditis, fibrosis and cardiomegaly) than those with high IFN-gamma and TNF-alpha levels. These animals produced low IL-10 levels in the culture supernatant and serum during the acute phase and did not produce IL-10 in the heart during the chronic phase of the disease. Our findings showed that Beagle dogs are a good model for studying the immunopathogenic mechanism of Chagas disease, since they reproduce the clinical and immunological findings described in chagasic patients. The data suggest that the development of the chronic cardiac form of the disease is related to a strong Th1 response during the acute phase of the disease, while the development of the indeterminate form results from a blend of Th1 and Th2 responses soon after infection, suggesting that the acute phase immune response is important for the genesis of chronic cardiac lesions.

Immunological imbalance between IFN-³ and IL-10 levels in the sera of patients with the cardiac form of Chagas disease

The immune response is crucial for protection against disease; however, immunological imbalances can lead to heart and digestive tract lesions in chagasic patients. Several studies have evaluated the cellular and humoral immune responses in chagasic patients in an attempt to correlate immunological findings with clinical forms of Chagas disease. Moreover, immunoglobulins and cytokines are important for parasitic control and are involved in lesion genesis. Here, cytokine and IgG isotype production were studied, using total epimastigote antigen on sera of chagasic patients with indeterminate (IND, n = 27) and cardiac (CARD, n = 16) forms of the disease. Samples from normal, uninfected individuals (NI, n = 30) were use as controls. The results showed that sera from both IND and CARD patients contained higher levels of Trypanosoma cruzi-specific IgG1 (IgG1) antibodies than sera from NI. No difference in IgG2 production levels was observed between NI, IND and CARD patients, nor was a difference in IL-10 and IFN-³ production detected in the sera of IND, CARD and NI patients. However, IND patients displayed a positive correlation between IL-10 and IFN-³ levels in serum, while CARD patients showed no such correlation, indicating an uncontrolled inflammatory response in CARD patients. These findings support the hypothesis that a lack of efficient regulation between IFN-³ and IL-10 productions in CARD patients may lead to cardiac immunopathology.

Trypanosoma cruzi: blood parasitism kinetics and their correlation with heart parasitism intensity during long-term infection of Beagle dogs

The goals of the present study were to evaluate the kinetics of blood parasitism by examination of fresh blood, blood culture (BC) and PCR assays and their correlation with heart parasitism during two years of infection in Beagle dogs inoculated with the Be-78, Y and ABC Trypanosoma cruzi strains. Our results showed that the parasite or its kDNA is easily detected during the acute phase in all infected animals. On the other hand, a reduced number of positive tests were verified during the chronic phase of the infection. The frequency of positive tests was correlated with T. cruzi strain. The percentage of positive BC and blood PCR performed in samples from animals inoculated with Be-78 and ABC strains were similar and significantly larger in relation to animals infected with the Y strain.Comparison of the positivity of PCR tests performed using blood and heart tissue samples obtained two years after infection showed two different patterns associated with the inoculated T. cruzi strain: (1) high PCR positivity for both blood and tissue was observed in animals infected with Be-78 or ABC strains; (2) lower and higher PCR positivity for the blood and tissue, respectively, was detected in animals infected with Y strains. These data suggest that the sensitivity of BC and blood PCR was T. cruzi strain dependent and, in contrast, the heart tissue PCR revealed higher sensitivity regardless of the parasite stock.

Trypanosoma cruzi high infectivity in vitro is related to cardiac lesions during long-term infection in Beagledogs

Trypanosoma cruzi is a hemoflagelate parasite associated with heart dysfunctions causing serious problems in Central and South America. Beagle dogs develop the symptoms of Chagas disease in humans, and could be an important experimental model for better understanding the immunopathogenic mechanisms involved in the chagasic infection. In the present study we investigated the relation among biological factors inherent to the parasite (trypomastigote polymorphism and in vitro infectivity) and immunoglobulin production, inflammation, and fibrosis in the heart of Beagle dogs infected with either T. cruzi Y or Berenice-78 strains. In vitro infectivity of Vero cells as well as the extension of cardiac lesions in infected Beagle was higher for Y strain when compared to Berenice-78 strain. These data suggested that in vitro infectivity assays may correlate with pathogenicity in vivo. In fact, animals infected with Y strain, which shows prevalence of slender forms and high infectivity in vitro, presented cardiomegaly, inflammation, and fibrosis in heart area. Concerning the immunoglobulin production, no statistically significant difference was observed for IgA, IgM or IgG levels among T. cruzi infected animals. However, IgA together IgM levels have shown to be a good marker for the acute phase of Chagas disease.

Expression and purification of functional, recombinant Trypanosoma cruzi complement regulatory protein.

The complement regulatory protein (CRP) of Trypanosoma cruzi is a developmentally regulated glycosylphosphatidylinositol (GPI)-anchored membrane protein that protects the parasite from complement-mediated killing, and is an important virulence determinant of the microorganism. CRP binds human complement components C3b and C4b to restrict activation of the complement cascade. Here, we report production of functional, recombinant T. cruzi CRP in mammalian cells and a one-step purification of the recombinant protein. Exchange of the crp DNA sequence encoding the carboxy-terminal GPI signal sequence with the corresponding sequence of decay accelerating factor (DAF) was necessary for recognition, cleavage, and addition of GPI in mammalian cells. CRP production was assessed in two mammalian cell lines with crp-daf gene expression driven by three different transcription control regions: Rous sarcoma virus long terminal repeat, cytomegalovirus (CMV) immediate early gene, and chicken beta-actin promoter/CMV enhancer. We present evidence that CRP produced in transfected Chinese hamster Ovary (CHO) cells was functional and protected the cells from complement-mediated lysis. To facilitate purification of the recombinant protein, a hexahistidyl tag was incorporated at 3(') end of the cDNA upstream of the GPI anchor addition sequence. An additional histidine fusion construct was made that allowed for secretion and recovery of recombinant protein from culture supernatant fluid. Both membrane and secreted forms of the protein were purified in one step by nickel nitrilotriacetic acid. The production and purification of functionally active CRP in a non-infectious expression system will allow for structure and function studies aimed at identifying the active site(s) of this protein.

Antígenos solúveis liberados por tripomastigotas de Trypanosoma cruzi utilizados no teste de Elisa para detectar cura em pacientes chagásicos após tratamento específico / Soluble antigens released by Trypanosoma cruzi trypomastigotes used Elisa to detect cure in chagasic patients following specific treatment

Dois antígenos solúveis de tripomastigotas do Trypanosoma cruzi, um obtido de sobrenadantes de culturas celulares (AgSb) e o outro excretado/secretado por essas formas em meio de cultura (AgES), foram avaliados em um teste de ELISA para o diagnóstico da infecçao chagásica e controle de cura de pacientes tratados. Os pacientes tratados apresentavam testes de lise mediada pelo complemento e hemoculturas repetidamente negativos, apesar de permanecerem com a sorologia convencional positiva (pacientes dissociados). O teste de lise negativo indica que estes pacientes eliminaram a infecçao. Entre os controles com infecçao ativa, os AgSb e os AgES detectaram respectivamente 93 e 100 por cento dos casos. No entanto, entre os pacientes dissociados, o teste de ELISA, utilizando os AgSb e AgES, foi positivo com 28 por cento e 5 por cento dos soros, respectivamente. Portanto, este teste com os AgES é indicado para o controle de cura da doença de Chagas, podendo vir a substituir a reaçao de lise mediada pelo complemento no acompanhamento sorológico individual de pacientes tratados.

Difference in susceptibility to lysis between clones of the Y strain of Trypanosoma cruzi

Three clones isolated from the Y strain of Trypanosoma cruzi--YP1, YP2 and YP3--were adapted to in vitro cultivation in VERO cells. The recovery of the parasites from the Y strain and clone YP3 was similar after 24 hr of contact with cells (3.2 per cent and 2.7 per cent , respectively) and much lower than the recovery of clones YP1 and YP2 (56.7 per cent and 60.0 per cent of inoculum, respectively). After five days incubation, the ratio Trypomastigotes/Amastigotes released into the supernatants was about 90/10 for clone YP1, YP3 and Y strain, and 50/50 for clone YP2. After nine days, the ratio was 62/38 for clone YP1, 97/3 for clone YP3, 81/19 for Y strain and 50/50 for clone YP2. The susceptibility of tissue culture derived trypomastigotes (TCT) to lysis in the presence of chronic chagasic human sera and human complement was assessed using Complement Mediated Lysis reaction (CML). Trypomastigotes from clone YP2 were consistently less susceptible to CML ( per cent lysis less than 20), than parasites from the other clones and Y strain. Parasites of clone YP3 had susceptibility to CML comparable to that of the Y strain (about 70 per cent ), while TCT of clone YP1 had intermediary susceptibility (40 per cent )

Criopreservacao de formas de cultura do Trypanosoma cruzi. / Cryopreservation of Trypanosoma cruzi culture form

Formas de cultura de diferentes cepas do T.cruzi foram submetidas a varios processos de criopreservacao. As percentagens de recuperacao, avaliadas pela motilidade dos parasitas, foram consideradas como adequadas com algumas das tecnicas empregadas, variando entre 60 a 80%. A estabilidade das caracteristicas biologicas do material criopreservado foi investigada atraves do estudo das curvas de crescimento e diferenciacao em meio acelular, infectividade para celulas de cultura de tecido ("Vero"), diferenciacao intracelular em cultura de tecido assim como infectividade e curso da infeccao em animais de laboratorio.De um modo geral essas caracteristicas nao foram significativamente alteradas no material congelado e estocado por diferentes periodos de tempo