Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Olivieri, B. P. Articles by Cotta-de-Almeida, V. Search for Related Content PubMed PubMed Citation Articles by Olivieri, B. P. Articles by Cotta-de-Almeida, V.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, May 2005, p. 1981-1987, Vol. 49, No. 5
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.5.1981-1987.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Benznidazole Therapy in Trypanosoma cruzi-Infected Mice Blocks Thymic Involution and Apoptosis of CD4⁺CD8⁺ Double-Positive Thymocytes

B. P. Olivieri,^1* D. A. Farias-De-Oliveira,² T. C. Araujo-Jorge,¹ and V. Cotta-de-Almeida¹

Laboratory of Cell Biology, Department of Ultrastructure and Cell Biology,¹ Laboratory on Thymus Research, Department of Immunology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil²

Received 13 July 2004/ Returned for modification 11 October 2004/ Accepted 27 January 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Several alterations involving peripheral lymphoid organs have been extensively described after experimental Trypanosoma cruzi infection. Thymic involution occurs as well in infected mice, with both structural and functional alterations in the organ. Despite these abnormalities, specific immune response proceeds to control parasitemia and the participation of T lymphocytes is essential. However, there are relatively few studies on the impact of benznidazole (N-benzyl-2-nitroimidazole acetamide) upon this response. In this present work, we decided to evaluate the impact of benznidazole treatment upon the thymus involution following acute T. cruzi infection in mice. We have provided evidence that benznidazole treatment controls the severe abnormalities seen in the thymus due to T. cruzi infection. The thymocyte loss related to the depletion of double-positive CD4⁺CD8⁺ thymocytes was clearly prevented, corroborating the idea that the mechanism responsible for the prevention of thymus involution is related to the decrease of apoptosis rate in this subset after benznidazole treatment. Furthermore, we demonstrated the prevention of enhanced extracellular matrix deposition in the thymus. In conclusion, the preservation of thymus homeostasis, even though partial, was accomplished after benznidazole treatment. Our data are consistent with the notion that different outcomes of T. cruzi infection may be linked to differences in the parasite load concomitant to fine tuning of the host immune response.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Chagas' disease affects 16 to 18 million people in the Americas, with more than 300,000 new cases every year (34). Human infection with the protozoan Trypanosoma cruzi results in an acute phase of variable duration characterized by detectable parasitemia and myocarditis (16), followed by a lifelong asymptomatic phase with scarce circulating parasites. About one-third of the infected individuals develop chronic forms of the disease represented by the chronic Chagasic cardiomyopathy and/or digestive problems, the latter in minor frequency (8). It is proposed that the pathogenesis of Chagasic cardiomyopathy includes parasite persistence and/or autoimmunity (32).

As part of experimental T. cruzi-infection, some typical alterations concerning the immune system are observed, such as polyclonal B and T lymphocyte activation in secondary lymphoid organs, hypergammaglobulinemia, and immunosuppression towards homologous and heterologous antigens (21, 22, 23). Despite these abnormalities, specific immune responses can proceed to control parasitemia, and the participation of T lymphocytes is essential in this process as well as in the inflammatory response (14, 22, 33). Both structural and functional alterations in the thymus are also hallmarks of acute T. cruzi infection, with a clear acute involution expressed by loss of absolute mass and reduction in the organ cellularity (17, 29). The disturbances in the thymic lymphoid compartment are related to a decrease in the numbers of immature CD4⁺CD8⁺ double-positive thymocytes, and a relative increase in the numbers of single-positive CD4⁺ or CD8⁺ cells. The existence of immature T lymphocytes bearing prohibited T-cell receptor variable ß segments that leave the thymus and then differentiate into mature CD4⁺ or CD8⁺ lymphocytes in the peripheral lymphoid organs (20) may contribute to the autoimmune process observed in later phases of infection. As well, alterations on the thymus microenvironment with densification of its epithelial network and an increase in the expression of extracellular matrix ligands and receptors were identified (6, 7, 29).

The specific treatment of Chagas' disease is based on the use of the nitroderivative benznidazole (N-benzyl-2-nitroimidazole acetamide), which is known to reduce parasitism, eliminate the acute-phase symptoms, and abbreviate the course of infection (4, 5). Despite the large number of studies focusing on the immune response following T. cruzi infection, there are relatively few studies on the impact of benznidazole treatment upon this response. In this present work, we evaluated the impact of benznidazole treatment upon the thymic changes following acute T. cruzi infection.

Top Abstract Introduction Materials and Methods Results Discussion References

 
T. cruzi infection. Male albino Swiss mice (age, 6 to 8 weeks; weight, 16 to 20 g) were kept under controlled conditions in our animal facilities. All procedures involving animal use followed the rules of the Animal Ethics Committee of the Oswaldo Cruz Foundation, protocol 99/01. Mice were inoculated by the intraperitoneal route with 200 µl of saline solution containing 10⁴ trypomastigotes of T. cruzi strain Y. The parasites were obtained from the blood of infected albino Swiss mice at the time of the parasitemia peak level by differential centrifugation steps.

Benznidazole treatment. Mice were submitted to ad libitum treatment with the addition of 0.25 mg of N-benzyl-2-nitroimidazole acetamide (benznidazole; Rochagan, Roche, Rio de Janeiro, Brazil) per milliliter in the drinking water, from days 7 to 21 after infection. The daily volume drunk by the mice was monitored for calculation of the total amount of drug consumed, which corresponded to 62.5 mg of benznidazole/kg/day, as described previously (25).

Experimental groups. Mice were separated into the following groups: Noninfected, nontreated mice; Noninfected, benznidazole-treated mice; T. cruzi-infected, nontreated mice; and T. cruzi-infected, benznidazole-treated mice.

Parasitological features. T. cruzi-infected mice had their parasitemia level evaluated at several times after infection. Succinctly, 5 µl of fresh blood obtained from the mouse tail were compressed between a glass slide and a coverslip (18 by 18 mm). The parasitemia level per milliliter was assessed after scoring the number of parasites in 50 fields and then this number was multiplied by a conversion factor, considering the number of microscopic fields in the area under specific magnification. The cumulative mortality was daily monitored until day 40 after infection.

Thymocyte phenotyping by flow cytometry. Following euthanasia, thymuses from three to four animals of each experimental group were collected at days 9 and 14 after the infection. Cell suspensions were gently obtained in a tissue grinder in RPMI medium supplemented with 5% fetal calf serum. Cell numbers and viability were determined by trypan blue dye-exclusion and counting in a hemacytometer. For thymocyte subsets cytofluorometric analysis, 10⁶ cells were placed in 96-well round-bottom plates and had their immunoglobulin Fc receptors blocked with the monoclonal antibody anti-FcRII/III (hybridoma 2.4G2 kindly provided by Marc Daëron, Institut Nationale de la Santé et de Recherche Médicale, Unité 255, Paris, France).

Following a 20-min incubation, the cells were centrifuged and suspended with appropriate dilutions of fluorescein isothiocyanate-labeled anti-CD4, phycoerythrin-labeled anti-CD8 (both purchased from Sigma Chemical Company, St. Louis, Mo.) and Tri-Color labeled anti-CD3 (Caltag Laboratories, San Francisco, Calif.) rat monoclonal antibodies. Isotype-matched labeled monoclonal antibodies were used as controls. After 20-min incubation, the cells were washed, fixed with formaldehyde 1% in phosphate buffered saline and stored at 4°C. Data acquisition of three-color staining was performed on a FACSCalibur flow cytometer (Becton Dickinson, San Jose, Calif.) equipped with the Cell Quest software (Becton Dickinson). Total 10,000 events were collected in a viable lymphocyte-enriched region defined according to forward- and side-scatter parameters, and further confirmed in some experiments with propidium iodide staining. The flow cytometry application software CellQuest (Becton Dickinson) or WinMDI2.8 (by Joseph Trotter, Scripps Research Institute, San Diego, CA) were employed for additional data analysis.

Thymocyte death analysis by flow cytometry. For the in vivo cell death detection and quantification studies, 10⁶ cells were incubated with monoclonal antibodies against CD4 and CD8 molecules as described above. After washings, these cells were incubated with 20 µg of 7-aminoactinomycin D (Sigma Chemical Company, St. Louis, Mo.) per milliliter for 20 min at 4°C, and immediately analyzed on the flow cytometer. A total 25,000 events were collected and separated in gates defining viable cells, early apoptotic cells and late apoptotic or necrotic cells, as previously described (30).

Immunofluorescence staining for confocal microscopy analysis. Thymuses from 3 to 4 animals of each experimental group were obtained at day 14 after infection, embedded in Tissue-Tek (Miles, Elkhart, IN) and preserved at –70°C for Confocal Microscopy analysis of extracellular matrix ligands. Four-µm thick cryostat sections were obtained (Cryostat Leica CM 1850, Leica Microsystems Inc., Buffalo, NY) and were immersion-fixed with acetone. Cryosections were then washed 3 times with phosphate buffered saline solution and the blockade of unspecific binding sites was performed with 1% bovine serum albumin at 4°C. Following 18 h incubation, sections were incubated with appropriate dilutions of rabbit antisera specific for mouse fibronectin or laminin, purchased from Institut Pasteur (Centre de Radioanalyse, Lyon, France) for 30 min at 37°C, and then washed three times with phosphate buffered saline solution. As developing system, appropriate dilutions of tetramethyl rhodamine isothiocyanate-labeled goat anti-rabbit immunoglobulin (Biosys, Compiegne, France) were added. Isotype-matched labeled monoclonal antibodies were used as controls. After washings, a coverslip was placed onto the glass slide and an antifading agent was added before data acquisition on the Confocal Microscopy (Carl Zeiss, Germany). Data analysis was carried out in Carl Zeiss LSM (Carl Zeiss, Germany) software and further image processing with Adobe Photoshop (Adobe Systems Inc., San Jose, Calif.).

Statistical analysis. Statistical analysis were performed according to the Mann-Whitney test, with values being considered significant when P < 0.05.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Herein, we illustrate results confirming that reduction of parasitic load after benznidazole therapy reduces parasitemia and mortality of T. cruzi-infected Swiss mice (1). As expected, T. cruzi infection in mice led to high parasitemia values, and the parasitemia peak was detected at day 9 after infection (Fig. 1A). Therapy with benznidazole employed from 7 to 21 days after infection led to a lower peak level of parasitemia, as well as early control of the circulating parasites (Fig. 1B) as formerly described (25). Such results are reinforced by the low mortality rate seen in treated mice compared to the lethal condition in the absence of trypanocidal treatment (Fig. 1B).

View larger version (19K): [in this window] [in a new window]

FIG. 1. Parasitemia and mortality reduction in Trypanosoma cruzi-infected mice after benznidazole therapy. Each point represents the individual data collected from eight mice per point of analysis of infected nontreated (I, circles) and infected benznidazole-treated mice (IBz, squares). (A) Parasitemia determination, with vertical bars representing the mean + standard deviation for each time point; (B) rate of cumulative mortality.

The thymus atrophy was very prominent in T. cruzi-infected nontreated mice, especially on day 14 after infection (Fig. 2). The reduction in the number of thymocytes was evident in infected nontreated mice from day 9 after infection, and intensified until day 14 of infection. In contrast, benznidazole treatment prevented the severe thymus atrophy seen in nontreated mice (Fig. 2). Even though the prevention of thymus involution was not complete in all benznidazole-treated mice, it was clearly attenuated.

View larger version (15K): [in this window] [in a new window]

FIG. 2. Attenuation of thymus involution in benznidazole-treated mice following Trypanosoma cruzi infection. Individual thymocyte numbers of noninfected (N, open circles), infected nontreated (I, solid circles), noninfected benznidazole-treated (NBz, open squares), and infected benznidazole-treated (IBz, solid squares) mice, at days 9 (A) and 14 (B) after infection. Horizontal bars represent mean values of three independent experiments, with a total of 8-12 mice/experimental group. * = different from N, ** = different from I (P < 0.05).

Cytofluorometric studies performed to determine the relative frequency of the subsets of thymocytes defined by CD3, CD4, and CD8 staining revealed clear changes in the composition of thymocyte subsets after benznidazole treatment in infected mice, especially at day 14 after infection, as illustrated in Fig. 3. A prominent CD4⁺CD8⁺ double-positive cell loss was detected in T. cruzi-infected nontreated mice (Fig. 3B), concomitant to a relative increase in the single-positive CD4⁺ and CD8⁺ subsets (Fig. 3B), and an up-regulation of CD3 expression (Fig. 3F). Altogether, these findings indicate a predominance of mature thymocytes in infected nontreated mice. Benznidazole therapy in T. cruzi-infected mice clearly reverted this condition (Fig. 3D and 3H), with mice displaying a thymocyte composition comparable to noninfected control mice (Fig. 3A, 3C, 3E, and 3G). The individual frequency of thymocyte subsets revealed that the phenomenon is present in all mice analyzed, but with slight individual variations (Fig. 3I, 3J, and 3K).

View larger version (35K): [in this window] [in a new window]

FIG. 3. Reduction of CD4+CD8+ double-positive thymocyte depletion in Trypanosoma cruzi-infected benznidazole-treated mice. Cytofluorometric studies of noninfected (N), infected nontreated (I), noninfected benznidazole-treated (NBz), and infected benznidazole-treated (IBz) groups at day 14 of infection, in a total of three independent experiments. A-D: Dot plots of CD4 and CD8 staining, with quadrants depicting the subsets identified by the phenotypic markers. E-H: Histograms of CD3 expression, with horizontal bars representing CD3low (M1) and CD3high (M2) expression, after the definition of positive staining by an immunoglobulin isotype control. Data are from one representative animal of each group. The numbers inside parentheses depict the relative frequency of each subset. Individual relative frequency of thymocyte subsets (I and J: single positive CD4+ or CD8+, K: double positive CD4+CD8+) defined by the phenotypic markers CD4 and CD8 in noninfected (N, open circles), infected nontreated (I, solid circles), noninfected benznidazole-treated (NBz, open squares), and infected benznidazole-treated (IBz, solid squares) mice. The solid horizontal bars represent the median values from three independent experiments, with a total of 7-12 mice/experimental group. * = different from N, ** = different from I (P < 0.05).

In order to understand the nature of thymocyte loss, cells were incubated with 7-aminoactinomycin D to determine the rate of apoptotic cell death. Total thymocyte staining indicated an apparent increase in the rate of apoptotic cell death in the infected nontreated group (Fig. 4B - marker), compared to noninfected and infected benznidazole-treated mice (Fig. 4A and 4D, marker). The determination of apoptotic death in CD4⁺, CD8⁺ and CD4⁺CD8⁺ double-positive subsets indicated that the increase in the total rate of apoptosis seen in infected nontreated mice (Fig. 4E) was due to the distinct increase of apoptosis in the CD4⁺CD8⁺ subset (Fig. 4F, 4G, and 4H). Notably, this effect was clearly blocked in T. cruzi-infected benznidazole-treated mice (Fig. 4E and 4H, IBz), which displayed a thymocyte subset composition more similar to noninfected mice (Fig. 4E and 4H, N and NBz). It is worth mentioning that benznidazole treatment in noninfected mice (NBz) did not induce significant changes in any aspect of the immune response studied (Fig. 2, 3, and 4, NBz).

View larger version (29K): [in this window] [in a new window]

FIG. 4. Benznidazole treatment prevented thymocyte loss in Trypanosoma cruzi infected mice. A-D: Cytofluorometric tridimensional graphs of relative cell number versus forward scatter (FSC) versus 7-aminoactinomycin D staining. Representation of total cell profile from noninfected (N), infected nontreated (I), noninfected benznidazole-treated (NBz), and infected benznizadole-treated (IBz) mice. Viable cells and early apoptotic cells are illustrated by left and right marks, respectively. E-F: Columns depict mean apoptotic cell rate in total, CD4+CD8+, CD4+ and CD8+ thymocytes from noninfected (N), infected nontreated (I), noninfected benznidazole-treated (NBz), and infected benznidazole-treated (IBz) mice, in three experiments with a total of 6-9 mice/experimental group. Vertical bars represent the 75th percentile values.

The expression of extracellular matrix molecules by immunofluorescence staining revealed the pattern of laminin expression in noninfected mice as a definite labeling at the basement membranes, in addition to a clear-cut expression at the medullary region of thymic lobules and a network of very thin fibres in the cortex (Fig. 5A and 5B). Noninfected benznidazole-treated mice (NBz) exhibited the same expression pattern (data not shown). In a different way, infected mice displayed profuse laminin deposition with the intensification of the network in the thymus parenchyma (Fig. 5C and 5D). Remarkably, benznidazole treatment in infected mice clearly prevented this deposition (Fig. 5E and 5F) with a laminin expression pattern equivalent to that observed in noninfected mice (Fig. 5A and 5B). Concerning fibronectin expression, similar results were found in the analyzed experimental groups (data not shown).

View larger version (92K): [in this window] [in a new window]

FIG. 5. Prevention of laminin deposition in the thymus of T. cruzi infected mice subjected to benznidazole therapy. Images of immunofluorescence staining and its representative topographic histograms from a representative animal of noninfected (A and B), infected nontreated (C and D) and infected benznidazole-treated (E and F) groups at day 14 after infection. The regions of thymus cortex (Co) and medulla (Me) are indicated.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Evidence has accumulated on the relevance of the thymus to host resistance after T. cruzi infection, where substantial immature thymocyte depletion has been well characterized (2, 6, 7, 17, 18, 20, 29). The infection is more severe, with higher parasitemia levels and increased mortality rate after thymus removal (31) or in athymic mice (11, 15), and accentuated myocardial lesions are related to adult thymectomy (3). More recent findings showed that mice susceptible to T. cruzi infection display significantly higher thymocyte loss compared to the resistant ones, with the latter showing recovery of the acute infection coinciding with thymic recovery (27). Our present results confirmed that benznidazole treatment of T. cruzi-infected mice increases the survival and promotes a reduction in the parasitic load. We also demonstrated for the first time an important prevention of thymus involution after benznidazole therapy in T. cruzi-infected mice and the apoptotic nature of this loss.

Our recent studies on thymus atrophy during T. cruzi infection supported a role for purinergic receptors on CD4⁺CD8⁺ T cells (19) and excluded FasL and perforin-mediated mechanisms for thymocyte cell death (12). In the present results, the clear prevention of thymocyte loss was associated with a subset profile more similar to the noninfected mice, where the majority of cells display an immature phenotype. These data support the new concept that the central lymphoid compartment is preserved in benznidazole-treated mice. This may be linked to the low mortality rate and better clinical condition seen in benznidazole-treated mice. In a recent work in the same experimental infection and therapy model, we demonstrated a different outcome concerning the host immune response on peripheral lymphoid tissues after benznidazole therapy (25). Following acute experimental infection, the expansion of CD8⁺ T lymphocytes with an effector/memory phenotype in-treated mice occurs concomitantly to the reduction of parasitemia and mortality (25). Accordingly, benznidazole treatment not only interrupted parasite replication but also induced new regulatory mechanisms, thus triggering a different host immune response.

Apoptosis is a common pathogenic mechanism used by parasites to evade the host immune response (9). As recently shown, T. cruzi appears to subvert the thymocyte fate through apoptosis induction, since an increased apoptosis rate in infected mice was identified as a cause for the significant cell depletion, with a suggestive involvement of the parasite trans-sialidase in this phenomenon (24, 26). Thymocyte apoptosis could be mediated by cytokines such as tumor necrosis factor alpha and nitric oxide (10, 13) since those mediators are present in higher levels when more pronounced CD4⁺CD8⁺ double-positive thymocyte loss occurs (27). Concurrently, amastigote forms of T. cruzi have already been identified in the thymus (29) and thymus cell number recovers after the control of parasitemia in later phases of infection (2, 18). According to these data, thymocyte loss is related to apoptosis induction, and the reduction of parasitic load promoted a recovery of the cell loss. As a result, the specific treatment of mice with benznidazole caused directly a decrease of the parasitic load, and thus we can assume that this reduction was associated with the lower apoptosis rate seen in these mice. As well, new regulatory mechanisms can figure in order to promote changes in the immune response such as the balance of cytokine levels.

The primary function of the thymus is to provide mature T cells for the peripheral lymphoid organs. In this process, bone marrow-derived precursors migrate, differentiate, and are selected through direct interaction with the thymic microenvironment, and the majority of the T-cell repertoire is thus originated. In a variety of infectious diseases, the thymus has been regarded as a target organ with disturbances in its microenvironment as well as in its lymphoid compartment. Cell migration is essential for intrathymic T-cell differentiation, and recent data suggest that chemokines and extracellular matrix elements are both important components involved in thymocyte migration (28). Abnormalities in thymocyte migration and differentiation were noticed in T. cruzi infection. Thymocyte depletion occurs along with the increased expression of extracellular matrix ligands and receptors, and an altered migratory capacity of immature T cells seems to correlate with a premature exportation of these cells (6, 7, 29). Our results demonstrated that the deposits of these extracellular matrix molecules were prevented in benznidazole treatment, suggesting that the defects in intrathymic cell trafficking in infected and treated mice might be inhibited in infected mice treated with benznidazole. The evaluation of such a proposition deserves further analysis.

In conclusion, benznidazole treatment was able to inhibit, although not completely, the severe abnormalities seen in the thymus following T. cruzi infection. Our results confirmed previous data that thymic involution is related to the depletion of double-positive CD4⁺CD8⁺ thymocytes and corroborate the idea that the mechanism responsible for the prevention of thymus involution after benznidazole treatment is related to the decrease of apoptosis rate in this subset. Prevention of enhanced extracellular matrix deposits suggests a regulation of the normal migration dynamics in the thymus after benznidazole therapy in infected mice. Therefore, our present results suggest that the preservation of thymus homeostasis might be contributing to the emergence of new regulatory mechanisms after trypanocidal treatment and possibly with an essential role in the increased survival observed in benznidazole-treated T. cruzi-infected mice.

 
This work was supported by PAPES, CAPES, and Oswaldo Cruz Institute.

We are grateful to Wilson Savino for interesting discussions, Elisngela Silva Monteiro for the help with data acquisition in the confocal microscope, and Linda Jelicks for reviewing the manuscript.

 
* Corresponding author. Mailing address: Laboratory of Cell Biology, Department of Ultrastructure and Cell Biology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Ave. Brasil 4365, Rio de Janeiro, Brazil CEP 21040-900. Phone: 5521 2598 4330. Fax: 5521 2260 4434. E-mail: olivieri{at}ioc.fiocruz.br.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Andrade, S. G., and R. M. Figueira. 1977. Estudo experimental sobre a ação terapêutica da droga Ro 7-1051 na infecção por diferentes cepas do Trypanosoma cruzi. Rev. Inst. Med. Trop. São Paulo. 19:335-341.[Medline]
2
2. Antunez, M. I., R. E, Feinstein, R. L. Cardoni, and K. O. Gronvik. 1997. Trypanosoma cruzi: T cell subpopulations in the Peyer's patches of BALB/c infected mice. Exp. Parasitol. 87:58-64.[CrossRef][Medline]
3
3. Bottasso, O. A., S. S. Revelli, H. Davila, J. L Valenti, O. C. Musso, M. E. Ferro, M. Romero-Piffiguer, and J. C. Morini. 1993. Enhanced myocardial lesions in chronically Trypanosoma cruzi-infected rats subjected to adult thymectomy. Immunol. Lett. 37:175-180.[CrossRef][Medline]
4
4. Brener, Z. 1979. Present status of chemotherapy and chemoprophylaxis in human trypanosomiasis in the Western Hemisphere. Pharmacol. Ther. 7:71-90.[CrossRef][Medline]
5
5. Cançado, J. R., and Z. Brener. 1979. Terapeutica, p. 362-424. In Z. Brener, and Z. Andrade (ed.), Trypanosoma cruzi e Doença de Chagas. Guanabara Koogan, São Paulo, Brazil.
6
6. Cotta-de-Almeida, V., A. Bonomo, D. A. Mendes-da-Cruz, I. Riederer, J. De Meis, K. R Lima-Quaresma, A. Vieira-de-Abreu, D. M Villa-Verde, and W. Savino. 2003. Trypanosoma cruzi infection modulates intrathymic contents of extracellular matrix ligants and receptors and alters thymocyte migration. Eur. J. Immunol. 33:2439-2448.[CrossRef][Medline]
7
7. Cotta-de-Almeida, V., A. L. Bertho, D. M. S. Villa-Verde, and W. Savino. 1997. Phenotypic and functional alterations of thymic nurse cells following acute Trypanosoma cruzi infection. Clin. Immunol. Immunopathol. 82:125-132.[CrossRef][Medline]
8
8. Dias, J. C. P., and J. R. Coura. 1997. Epidemiologia, p. 33-65. In J. C. P. Dias, and J. R. Coura (ed.), Clínica e terapêutica da doença de Chagas, uma abordagem prática para o clínico geral. Fiocruz, Rio de Janeiro, Brazil.
9
9. DosReis, G. A., and M. A. Barcinski. 2001. Apoptosis and parasitism: from the parasite to the host immune response. Adv. Parasitol. 49:133-161.[Medline]
10
10. Fehsel, K., K. D. Kroncke, K. L. Meyer, H. Huber, V. Wahn, and V. Kolb-Vachofen. 1995. Nitric oxide induces apoptosis in mouse thymocytes. J. Immunol. 155:2858-2865.[Abstract]
11
11. Gonçalves-da-Costa, S. C., P. H. Lagrange, B. Hurtrel, I. Keer, and A. Alencar. 1984. Role of T lymphocytes in the resistance and immunopathology of experimental Chagas'disease. 1. Histopathological studies. Ann. Inst. Pasteur (Immunol.) 135:317-332.
12
12. Henriques-Pons, A., J. DeMeis, V. Cotta-De-Almeida, W. Savino, and T. C. Araujo-Jorge. 2004. Fas and perforin are not required for thymus atrophy induced by Trypanosoma cruzi infection. Exp. Parasitol. 107:1-4.[CrossRef][Medline]
13
13. Hernandez, C. T., and O. Stutman. 1993. Immune functions of tumour necrosis factor. I. Tumour necrosis factor induces apoptosis of mouse thymocytes and can also stimulate or inhibit IL-6-induced proliferation depending on the concentration of mitogenic stimulation. J. Immunol. 151:3999-4012.[Abstract]
14
14. Kierszenbaum, F. 1995. What are T-cell subpopulations really doing in Chagas' disease? Parasitol. Today 11:6-7.[CrossRef][Medline]
15
15. Kierszenbaum, F., and M. M. Pienkowski. 1979. Thymus-dependent control of host defense mechanisms against Trypanosoma cruzi infection. Infect. Immun. 24:117-120.[Abstract/Free Full Text]
16
16. Laguens, R. P., M. I. Argel, J. Chambo, R. Storino, and P. M. Cabeza-Meckert. 1994. Presence of antiheart and antiskeletal muscle glycolipid autoantibodies in the sera of patients with chagasic cardiopathy. Can. J. Cardiol. 10:769-776.[Medline]
17
17. Leite-de-Moraes, M. C., M. Hontebeyrie-Joskowicz, F. Leboulenger, W. Savino, M. Dardenne, and F. Lepault. 1991. Studies on the thymus in Chagas' disease. II. Thymocyte subset fluctuations in Trypanosoma cruzi-infected mice: relationship to stress. Scand. J. Immunol. 33:267-275.[CrossRef][Medline]
18
18. Leite-de-Moraes, M. C., M. Hontebeyrie-Joskowicz, M. Dardenne, and W. Savino. 1992. Modulation of thymocyte subsets during acute and chronic phases of experimental Trypanosoma cruzi infection. Immunol. 77:95-98.[Medline]
19
19. Mantuano-Barradas, M., A. Henriques-Pons, T. C. Araujo-Jorge, F. Di Virgilio, R. Coutinho-Silva, and P. M. Persechini. 2003. Extracellular ATP induces cell death in CD4+/CD8+ double-positive thymocytes in mice infected with Trypanosoma cruzi. Microbes Infect. 5:1363-1371.[CrossRef][Medline]
20
20. Mendes-da-Cruz, D. A., J. de Meis, V. Cotta-de-Almeida, and W. Savino. 2003. Experimental Trypanosoma cruzi infection alters the shaping of the central and peripheral T-cell repertoire. Microbes Infect. 5:825-832.[CrossRef][Medline]
21
21. Minoprio, P., A. Coutinho, M. Joskowicz, M. R. D'Imperio-Lima, and H. Eisen. 1986. Polyclonal lymphocyte responses to murine Trypanosoma cruzi infection. II. Cytotoxic T lymphocytes. Scand. J. Immunol. 24:669-679.[CrossRef][Medline]
22
22. Minoprio, P., H. Eisen, L. Forni, M. R. D'Imperio-Lima, M. Joskowicz, and A. Coutinho. 1986. Polyclonal lymphocyte responses to murine Trypanosoma cruzi infection. I. Quantitation of both T- and B-cell responses. Scand. J. Immunol. 24:661-668.[CrossRef][Medline]
23
23. Minoprio, P., S. Itohara, C. Heusser, S. Tonegawa, and A. Coutinho. 1989. Immunobiology of murine Trypanosoma cruzi infection: the predominance of parasite-nonspecific responses and the activation of TCRI T cells. Immunol. Rev. 112:183-207.[CrossRef][Medline]
24
24. Mucci, J., A. Hidalgo, E. Mocetti, P. F. Argibay, M. S. Leguizamón, and O. Campetella. 2002. Thymocyte depletion in Trypanosoma cruzi infection is mediated by trans-sialidase-induced apoptosis on nurse cells complex. Proc. Natl. Acad. Sci. USA. 99:3896-3901.[Abstract/Free Full Text]
25
25. Olivieri, B. P., V. Cotta-de-Almeida, and T. C. Araujo-Jorge. 2002. Benznidazole treatment following acute Trypanosoma cruzi infection triggers CD8⁺ T cell expansion and promotes resistance to reinfection. Antimicrob. Agents Chemother. 46:3790-3796.[Abstract/Free Full Text]
26
26. Risso, M. G., G. B. Garbarino, E. Mocetti, O. Campetella, S. M. Gonzalez Cappa, C. A. Buscaglia, and M. S. Leguizamon. 2004. Differential expression of a virulence factor, the trans-sialidase, by the main Trypanosoma cruzi phylogenetic lineages. J. Infect. Dis. 189:2250-2259.[CrossRef][Medline]
27
27. Roggero, E., A. Perez, M. Tamae-Kakazu, I. Piazzon, I. Nepomnachy, J. Wietzerbin, E. Serra, S. Revelli, and O. Bottasso. 2002. Differential susceptibility to acute Trypanosoma cruzi infection in BALB/c and C57BL/6 mice is not associated with distinct parasite load but cytokine abnormalities. Clin. Exp. Immunol. 128:421-428.[CrossRef][Medline]
28
28. Savino, W., D. A. Mendes-da-Cruz, J. S. Silva, M. Dardenne, and V. Cotta-de-Almeida. 2002. Intrathymic T-cell migration: a combinatorial interplay of extracellular matrix and chemokines. Trends Immunol. 23:305-313.[CrossRef][Medline]
29
29. Savino, W., M. C. Leite-de-Moraes, M. Hontebeyrie-Joskowicz, F. Leboulenger, and M. Dardenne. 1989. Studies on the thymus in Chagas'disease. I. Changes in the thymic microenvironment in mice acutely infected with Trypanosoma cruzi. Eur. J. Immunol. 19:1727-1733.[Medline]
30
30. Schmid, I., C. H. Uittenbogaart, B. Keld, and J. V. Giorgi. 1994. A rapid method for measuring apoptosis and dual-color immunofluorescence by single laser flow cytometry. J. Immunol. Methods. 170:145-157.[CrossRef][Medline]
31
31. Schmunis, G. A., S. M. G. Cappa, O. C. Traversa, J. F. Jaanovsky. 1971. The effects of immuno-depression due to neonatal thymectomy on infections with Trypanosoma cruzi in mice. Trans. R. Soc. Trop. Med. Hyg. 65:89-94.[CrossRef][Medline]
32
32. Soares, M. B. P., L. Pontes-de-Carvalho, and R. Ribeiro-dos-Santos. 2001. The pathogenesis or Chagas' disease: when autoimmune and parasite-specific immune response meet. An. Acad. Bras. Cienc. 73:547-559.[Medline]
33
33. Tarleton, R. L. 1995. The role of T cells in Trypanosoma cruzi infection. Parasitol. Today 1:7-9.
34
34. World Health Organization. 2002. Chagas disease. Tropical Disease Research, 18th Program Report, UNDP/WB/TDR, Geneva.

---

Antimicrobial Agents and Chemotherapy, May 2005, p. 1981-1987, Vol. 49, No. 5
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.5.1981-1987.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Olivieri, B. P. Articles by Cotta-de-Almeida, V. Search for Related Content PubMed PubMed Citation Articles by Olivieri, B. P. Articles by Cotta-de-Almeida, V.