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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the acute phase, trypomastigotes are easily detectable in the peripheral blood.1 Two to three weeks after infection with T. cruzi, circulating antibodies can be measured that correlates with a rapid decrease in parasite numbers. During the chronic phase of the infection, it becomes difficult to detect T. cruzi.
Trypanosoma cruzi has a single mitochondrion that contains a network of circular concatenated DNA, which is made up of thousands of maxicircles and minicircles. This kinetoplast DNA (kDNA) was used as target to detect T. cruzi in blood and tissue samples.2–4 The few minicircles so far sequenced showed a common organization.2 Each of them, is composed of four conserved regions, the so called minirepeats, which are interspersed by highly variable regions (VR) that have only a low degree of homology. Parasitic DNA and human genomic DNA are easily distinguishable because T. cruzi minicircle kDNA is characterized by an unequal A:C:G:T ratio, with less than 10% of the nucleotides being C.2
The scarcity of the parasite in the chronic phase suggests that autoimmune processes are involved in the pathology of Chagas’ disease.5,6 An autoimmune response may be initiated by T cells that are activated by T. cruzi antigens that closely resemble self-antigens.7 Alternatively, the parasite may cause tissue damage that leads to the exposition of cryptic host antigens and subsequently to the induction of the inflammation.1 In addition, antibodies specific for both T. cruzi antigens and self-peptides are present in the sera of patients with chronic Chagas’ heart disease.8,9 Autoreactive antibodies, such as those binding to the ß-adrenergic receptor, may interfere with the functional activity of the heart.10–12
To study the question of whether in the chronic phase of Chagas’ heart disease T. cruzi is present in the heart tissue of chagasic patients, we have used micromanipulation together with a polymerase chain reaction (PCR) and in situ hybridization (ISH). Cryosections were prepared from the heart tissue of three patients with chronic Chagas’ heart disease and labeled with antibodies specific for mononuclear cells. Combining immunohistology with a molecular biochemical analysis allowed us to correlate the degree of inflammation with the presence of T. cruzi. Amplification of both T. cruzi kDNA and nuclear DNA suggests that T. cruzi DNA is widespread in the heart tissue of patients with chronic Chagas’ heart disease. However, it appears that only remnants of T. cruzi DNA are present, rather than intact whole parasites. This interpretation was confirmed by ISH.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Patients and tissue samples.
Myocardial tissue samples were obtained from three patients with chronic Chagas’ heart disease who had active myocarditis with severe left ventricular dysfunction. Informed consent was obtained from all patients before participating in the study, and the project was approved by the clinical review board of the Instituto Cardiologia y Cirugia Cardiovascular, Fundacion Favaloro (Buenos Aires, Argentina). The impaired heart function was assessed by the measurement of the left ventricular diastolic diameter, the systolic function, and the left ventricular ejection fraction (Table 1
). The patients showed marked limitation of activity (class III-IV) by the New York Heart Association classification (Table 1).
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All patients were serologically positive for T. cruzi. Although the exact time of infection with T. cruzi is not known, it is most likely that the patients were infected in their childhood, which is in all cases more than 30 years ago. Patients did not receive medication to treat the infection with T. cruzi.
Staining of human tissue sections. Heart tissue samples were shock frozen and 8-µm cryosections were prepared.13 Frozen sections were fixed with acetone and stored at -70°C until used. To determine the degree of inflammation, consecutive sections of human heart tissue were single labeled with mouse monoclonal antibodies specific for human B cells (anti-CD20; Dakopatts, Roskilde, Denmark), plasma cells (Wue-1),13 follicular dendritic cells (Wue-2),13 T cells (anti-CD3, anti-CD4, and anti-CD8; Dakopatts), macrophages (anti-CD14 and anti-MAC-3; Dakopatts), followed by rabbit anti-mouse immunoglobulin (Dakopatts) and alkaline phosphatase-anti-alkaline phosphatase (APAAP) complex (Dakopatts). Immune complexes containing APAAP were detected by incubation with the a fuchsin substrate (Dakopatts). The nuclei of the cells were counterstained with Papanicolau hematoxylin.
Isolation of cells and preparation of DNA. Human tissue sections were labeled with antibodies specific for mono-nuclear cells. Single macrophages or small areas of approximately 100 heart cells were dissected using a micromanipulator (Narishige, Tokyo, Japan).13 The isolated cells were digested with proteinase K (0.7 mg/mL) for 1 hour at 50°C (Boehringer, Mannheim, Germany). The enzyme was inactivated by heating at 95°C for 10 minutes.
Amplification of T. cruzi minicircle kDNA and nuclear DNA by PCR. Parasite DNA was amplified by a nested PCR. For the primary amplification of minicircle kDNA, primers specific for the conserved region within the minirepeats of the T. cruzi minicircles were used. After 35 cycles of amplification with external primers S35 and S36,14 a 1-µL aliquot was taken and reamplified for 40 cycles using internal primers (S35-N, 5'-AKTTGAACGCCCCTCCCA and S36-N, 5'-ATTG-GGGTTGGTGTAATATAG-3'). The amplification program consisted of an initial denaturation step at 95°C for nine minutes, followed by cycles of denaturation at 94°C for one minute, annealing at 53°C for one minute, and extension at 72°C for one minute. The amplification mixture contained 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 0.1 mM MgCl2, 200 mM of each dNTP, 5 pmoles of each primer for the first amplification and 10 pmoles of each primer for the second amplification, and two units of ampliTaq Gold (Perkin Elmer, Boston, MA). Amplification resulted in a DNA fragment of approximately 270 base pairs that covers the VR sequence, in addition to a few nucleotides of the minirepeat. For the amplification of nuclear DNA, primers specific for a short interspersed repetitive element (SIRE) found in the genome of T. cruzi were used (SIRE A, 5'-GA GAGCTGGCTAACTTAAT-3' and SIRE B, 5'-TAM TTAMGAAGTGGCAGACT-3' and the nested primers SIRE A', 5'-GTATGAATCTTTTGGGAAGAAC-3' and SIRE B', 5'-GGTCCTCCAACCACAAGAC-3').15 The amplification was performed as described for kDNA.
Detection of T. cruzi by ISH. For ISH, a 120-base pair fragment was amplified using primers corresponding to a conserved region on the T. cruzi minicircle DNA (5'-GGTTTT-GGGAGGGGCGTTC-3' and 5'-ACACCAACCCCAATC-GAACC-3'). The probe was labeled with digoxigenin-UTP (Boehringer). To rule out that a negative result was due to sequence diversity in the minirepeat sequence of different strains of T. cruzi, three different probes were prepared from kDNA of T. cruzi isolated from the Tulahuen strain (probe A), from the heart tissue of patient 2 (probe B), and from the heart tissue of patient 3 Ci probe C). The sections were fixed with 4% paraformaldehyde (pH 7) for 20 minutes, treated with 0.2 N HCl for 20 minutes at room temperature, and digested with pronase (0.125 mg/mL) for 10 min at 37°C. The pronase was inactivated for 30 seconds with 0.1 M glycine. To reduce the background, sections were washed with triethanolamine (pH 8) for 10 min and were fixed again with 4% paraformaldehyde for 20 min. The ISH was performed after dehydration of the samples with a graded ethanol series. A 50-µL hybridization solution containing 50% deionized formamide, 0.3 M NaCl, 10 mM Tris-HCl (pH 7.5), 10 mM sodium phosphate (pH 6.8), 5 mM EDTA, 1x Denhardt’s solution (0.02% polyvinylpyrrolidone, 0.02% Ficoll, 0.02% bovine serum albumin), 250 µg/mL of sonicated salmon sperm DNA, and 10% dextran sulfate was used and contained the digoxigenin-labeled probe. After an 18-hour hybridization at 45°C, slides were washed for 15 min in 2x SSC (150 mM sodium chloride, 15 mM sodium citrate, pH 7.0) at room temperature and for 15 min in 2x SSC at 37°C. The hybridization signal was detected by incubating slides with an anti-digoxigenin-specific antibody conjugated with alkaline phosphatase (Boehringer).
Sequence analysis. Sequences were determined from minicircle DNA and nuclear DNA amplified from T. cruzi DNA. For sequence analysis, amplified PCR products were cloned into the pCR II vector with the TA cloning system, version 2.3 (Invitrogen, Carlsbad, CA). Inserts were sequenced from both sides by using a -40 and +40 digoxigenin end-labeled primer. Sequence reaction products were applied to a direct blotting machine (MWG, Ebersberg, Germany) and run onto a Nylonbind membrane (Serva, Mannheim, Germany) as described previously.13 Some of the clones were sequenced using the BigDye terminator cycle sequencing ready reaction kit (Perkin Elmer) and analyzed with the automatic ABI PRISM 310 Genetic Analyzer (Perkin Elmer).
The sequences derived from parasitic DNA amplifications were analyzed by using the Blast search program Gene bank database (National Center for Biotechnology Information, National Institutes, of Health, Bethesda, MD).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The biopsy sample of patient 2 showed a higher degree of inflammation. Here, small clusters of up to 30 mononuclear cells were found between the myocytes (Figure 1b). In an area of approximately 6 mm2, 3–5 small foci of such cells were seen. Labeling with a monoclonal antibody specific for the different mononuclear cells showed that these infiltrates were composed mainly of T cells. A few macrophages and plasma cells were often seen near the T cells (moderate inflammation). The heart tissue from transplant patient 3 showed severe signs of inflammation. The myocardial tissue was disrupted and large spaces had formed between the myocardial cells. In addition to fibrotic tissue, a strong infiltration of lymphoid cells was observed (Figure 1c). Although some parts of the analyzed heart tissue were more heavily infiltrated than others, there was practically no area without signs of inflammation. Specific labeling showed that some of these cell clusters contained more than 1,000 CD8+ and CD4+ T cells. Groups of plasma cells were occasionally found within such large T cell clusters. In addition, individual plasma cells were found dispersed throughout the tissue.
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). The parasite was spread throughout the heart tissue and small nests of T. cruzi amastigotes were easily detectable (Figure 2a and b). This was different at the chronic phase of infection. One hundred eighty days after infection with T. cruzi, there was little evidence of the parasite in the murine heart tissue. The only positive hybridization signals were seen on two consecutive sections, and these were in the same area of the heart tissue (Table 2 and Figure 2c).
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). Probes were generated from kDNA of the Tulahuen strain, and the kDNA isolated from the heart tissue of patients 2 and 3. Nevertheless, all sections from patients 2 and 3 tested were negative for T. cruzi. Although all three patients were serologically positive for T. cruzi, microscopic analysis of heart tissue sections failed to detect nests of amastigotes. The only positive hybridization signal was seen in the heart tissue of patient 1 (Table 2 and Figure 2d). Since the signal was detected in the same area of three consecutive sections, it suggested the presence of a few T. cruzi amastigotes in one of the heart cells. Presence of parasitic DNA in the heart tissue of patients with chronic Chagas’ heart disease. To test for the presence of T. cruzi in heart tissue, small regions of heart tissue were dissected with a micromanipulator, DNA was extracted, and the VR sequences of the minicircle kDNA were amplified and sequenced.
Eighteen independent PCRs were performed from the biopsy sample of patient 1, which showed only a mild degree of inflammation (Table 3). Cells were dissected only from those areas where small groups of mononuclear cells were seen. No PCR product was detected in any of the amplifications after 35 cycles. Only after 35 additional cycles did 39% of the amplifications give a positive signal (Table 3). In addition, no more than two different VR sequences were isolated (Table 4). from each of the PCR amplifications. These data suggest that T. cruzi DNA is present in the heart tissue of patients with chronic Chagas’ heart disease. However, when one considers the abundance of minicircle DNA in the single mitochondrion, there seems to be only traces of T. cruzi DNA in the heart tissue.
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and 4). A similar analysis was performed with the tissue of patient 3. Only one of 16 amplifications resulted in a positive PCR signal and only one additional VR sequence was isolated (Tables 3 and 4). Despite the higher degree of inflammation, T. cruzi kDNA was amplified less frequently than from the heart tissue of patient 1.
The presence of parasite DNA in the heart tissue of patients with chronic Chagas’ heart disease was further substantiated by the amplification of SIRE, a middle repetitive element of the nuclear genome of T. cruzi (Table 3).15,16 Again, two cycles of PCR amplification were necessary to obtain a positive signal. To confirm the presence of the SIRE sequence in the analyzed heart tissue, the PCR product of two of the amplifications were cloned and sequenced (Figure 3).
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). Two of 11 macrophages gave a positive PCR signal; however, in both cases, 70 cycles of amplification were required. Sequence analysis showed that in both cases a single VR region was amplified (sequences VR 1.6 and VR 1.7, Table 4). These results support the interpretation that at the chronic phase of Chagas’ heart disease, macrophages present in the heart tissue contain parasitic DNA.
Presence of T. cruzi kinetoplast DNA in murine heart tissue.
When murine sections from the chronic phase of infection with T. cruzi were analyzed, similar to the results obtained with human heart tissue, no positive signal was obtained after 35 cycles of amplification (Table 5). In contrast, when heart tissue from a mouse during the acute phase of infection with T. cruzi, was analyzed, 35 cycles were sufficient to obtain a strong signal in the PCR. The presence of both trypanosomal kDNA and nuclear DNA (SIRE sequences) was demonstrated (Table 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Indeed, we found little evidence for the persistence of T. cruzi parasites in chronically infected murine heart tissue. Similarly, in the heart samples from patients 2 and 3 there was no evidence of T. cruzi. Only in the sample from patient 1, in one area of the 14 sections analyzed, a single heart cell was found that seemed to carry T. cruzi amastigotes (Figure 2).
Nevertheless, when a more sensitive method was used, T. cruzi DNA could be detected in all three human tissue samples tested (Table 4). This supports previous results that T. cruzi DNA is widespread in the heart tissue of patients with chronic Chagas’ heart disease.17–19 Although the isolated T. cruzi kDNA sequences showed a low degree of homology with published VR sequences, they are clearly of T. cruzi origin. Amplification with kDNA-specific primers resulted in fragments of the expected length of 270–280 nucleotides. In addition, all sequences showed the under representation of cytidines characteristic for T. cruzi kDNA (Table 4).2
Some studies have suggested a positive correlation between the degree of inflammation and the presence of T. cruzi DNA in the heart tissue.17,20 However, we found by ISH no evidence for the presence of intact T. cruzi parasites in inflamed areas. In particular, in heart tissue sections of patient 3 containing multiple macrophages, T cells, and plasma cells (Figure 1c), no T. cruzi amastigotes were detected.
When one considers that in a single T. cruzi mitochondrion there are more than 1,000 copies of minicircle DNA, it was surprising that in contrast to the acute phase, 35 PCR cycles were not sufficient to obtain an amplification product, and that none of the amplifications from the chronically infected tissue of patients or mouse resulted in more than two different VR sequences. It seems unlikely that there are intact mitochondrial minicircle networks in the inflamed heart tissue. Rather, it suggests that the analyzed heart tissue samples contain mostly remnants of T. cruzi DNA. This interpretation is supported by the amplification of SIRE, a repetitive sequence of the nuclear DNA. Again, two rounds of amplification were necessary to obtain a positive signal.
In agreement with a number of studies,19,21,22 there is a low prevalence of intact T. cruzi parasites in the chronically inflamed tissue. However, T. cruzi DNA appears to be widespread. One interpretation might be that traces of T. cruzi DNA remain in the heart tissue after the initial parasite infection. Vaccination experiments have shown that DNA injected into muscle cells can persist for months.23 Thus, the PCR signals may derive from DNA that persists in the absence of living parasite.
There is evidence that even years after infection living parasites are present in chronic chagasic patients. For example, treatment with benznidazole or other anti-parasitic drugs reduces the titer of T. cruzi-specific antibodies, suggesting that surviving T. cruzi are the cause of continuous parasite-specific antibody titers in the serum of these patients.24 Furthermore, the finding of infection with T. cruzi after transfusion with blood from chronic chagasic donors and the observation that there is reactivation of parasitemia in immuno-suppressed patients supports the interpretation that T. cruzi parasites persist in the host.25,26 However, this study suggests that it is unlikely that the heart tissue of patients with chronic Chagas’ heart disease is a major reservoir of the parasites. Both ISH and PCR demonstrate that if intact T. cruzi is present in the chronically inflamed tissue, then the numbers involved are vanishingly small.
Received June 12, 2002. Accepted for publication October 28, 2002.
Acknowledgments: We thank Dr. Meyer zum Büschenfeld and Dr. M. Hontebeyrie for the generous gift of murine heart tissues infected with T. cruzi, and Sonia Lafon for technical assistance.
Financial support: This work was supported by the European Union EU program (ARG/B7-3011/94/28A), and partially supported by the World Health Organization Special Program for Research and Training in Tropical Diseases; the International Atomic Energy Agency; the University of Buenos Aires (grant ANPC-FONCYT 05-802, SECyT); Beca Ramón Carrillo (Ministry of Health of Argentina); and the Bunge y Born Foundation of Argentina. Mariano J. Levin was supported in part by an International Research Scholar Grant from the Howard Hughes Medical Institute (Chevy Chase, MD). The Deutsche Rheuma ForschungsZentrum is supported by the Berlin Senate of Research and Education.
Authors’ addresses: Fernando E. Elias, Genethor GmbH, Robert-Rössle-Strasse 10, D-13125 Berlin, Germany, Telephone: 49-30-9489 2515, Fax: 49-30-9489 2516, E-mail: f.elias{at}gmx.net. Carlos A. Vigliano and Rubén P. Laguens, Departamento de Anatomía Pa-tológica, Instituto de Cardiología y Cirugía Cardiovascular, Fundación Favaloro, Solís 453, 1428 Buenos Aires, Argentina, Telephone: 54-114-378-1315. Mariano J. Levin, Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, Vuelta de Obligado 2490, 1428 Buenos Aires, Argentina, Telephone: 54-11-4786-5516, Fax: 54-11-4786-8578, E-mail: mlevin{at}dna.uba.ar. Claudia Berek, Deutsches Rheuma-ForschungsZentrum, Schumannstrasse 20/21, 10117 Berlin, Germany, Telephone: 49-30-28460 711, Fax: 49-30-28460 712, E-mail: berek{at}drfz.de.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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