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Antiproliferative effect of sera from chagasic patients on Trypanosoma cruzi epimastigotes. Involvement of xanthine oxidase
Acta Tropica 109 (2009) 219–225
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Antiproliferative effect of sera from chagasic patients on Trypanosoma cruzi epimastigotes. Involvement of xanthine oxidase Susana M. Hernandez a , Rodolfo A. Kolliker-Frers b , Marcela S. Sanchez c , Gabriela Razzitte b , Rodny D. Britos a , Maria E. Fuentes a , Martha N. Schwarcz de Tarlovsky a,∗ a b c
School of Medicine, CAECIS, Universidad Abierta Interamericana, Montes de Oca 745, 1270 Buenos Aires, Argentina Laboratory of Parasitology, Jose Maria Ramos Mejia Hospital, Urquiza 609, 1211 Buenos Aires, Argentina Ciclo Básico Común, Universidad de Buenos Aires, Paraguay 2155, 1121 Buenos Aires, Argentina
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Article history: Received 20 August 2008 Received in revised form 10 November 2008 Accepted 14 November 2008 Available online 25 November 2008 Keywords: Trypanosoma cruzi Chagas disease Xanthine oxidase Reactive oxygen species Hydrogen peroxide
a b s t r a c t Serum from asymptomatic or symptomatic (with cardiovascular manifestations) chagasic patients depleted of the complement system displayed an antiproliferative effect on Trypanosoma cruzi epimastigotes, RA strain, when added to the growth medium. This effect was also observed when patient’s serum was depleted of specific antibodies. The antiproliferative effect was both time and concentration dependent. It was confined to the nondialyzable, high molecular weight, fraction of the serum. This effect was abrogated by allopurinol and catalase, and enhanced by N-ethylmaleimide. Xanthine oxidoreductase and xanthine oxidase activities were increased in the chagasic sera, while catalase activity remained unchanged. Parasites exposed to chagasic sera showed a decrease in Fe/superoxide dismutase activity as well as an increase in membrane lipoperoxidation. Our data provides evidence to support the idea that the antiproliferative activity observed in sera from chagasic patients may be due, at least partially, to a direct effect of hydrogen peroxide on the epimastigotes of T. cruzi. The increase of hydrogen peroxide to antiproliferative levels might result from an increase in xanthine oxidase activity in the serum of patients infected with the parasite. © 2009 Published by Elsevier B.V.
1. Introduction The trypanosomiases are a group of diseases affecting humans and livestock in Africa, Asia and South America. These diseases are caused by the infection of blood-dwelling protozoan parasites called Trypanosoma. Trypanosoma brucei subspecies T. brucei gambiense and T. brucei rhodesiense are the etiologic agents of sleeping sickness (African trypanosomiasis) while T. cruzi is the causative agent of Chagas disease (American Trypanosomiasis). Both parasites developed the genetic variability needed for survival in their host, by using different strategies. T. brucei changes periodically the expression of a group of variant surface glycoproteins (Cross, 1990; Vanhamme and Pays, 2004), whereas T. cruzi counts on the extreme heterogeneity of their population. The source of T. cruzi antigenic diversity may arise from metabolic changes in the mismatch repair pathway (Machado et al., 2006). Nevertheless human blood, unlike the blood of other mammals, has an efficient trypanolytic activity against Trypanosoma brucei brucei. This is due to the presence of non-immune serum factors that lyse the invading trypanosome. One lytic factor of human serum
∗ Corresponding author. Tel.: +54 11 4301 5240; fax: +54 11 4301 5240x107. E-mail address: [email protected] (M.N. Schwarcz de Tarlovsky). 0001-706X/$ – see front matter © 2009 Published by Elsevier B.V. doi:10.1016/j.actatropica.2008.11.013
against T. brucei brucei was characterized as a subset of high density lipoproteins (HDL) which invariably include haptoglobin related protein and apolipoprotein L1 (Raper et al., 1999; Drain et al., 2001; Pays et al., 2006). On the other hand, it has been observed that only Cape buffalo, among a wide variety of domestic animals, is resistant to infection by African trypanosomes. This resistance is due to accumulation of a trypanocydal concentration of hydrogen peroxide in the buffalo serum, during catabolism of xanthine by xanthine oxidase as a consequence of the infection (Muranjan et al., 1997; Black et al., 2001). Infection, as well as various forms of tissue damage, induces inflammatory reactions as an important part of innate immunity. The inflammatory reaction results in the expression of a number of cytokines. Among these cytokines, gamma-interferon (IFN␥), alpha-interferon (IFN-␣), tumor necrosis factor-alpha (TNF-␣), interleukin-1 (IL-1) and interleukin-3 (IL-3) stimulate xanthine oxidoreductase (XOR) expression (Berry and Hare, 2004). XOR is a housekeeping enzyme with a role in purine catabolism, detoxification and the regulation of the cellular redox potential. The XOR enzyme is a homodimer composed of catalytically independent subunits with an approximate molecular mass of 150 kDa each. It exists in two inter-convertible enzymatic forms, as xanthine dehydrogenase (XDH) (E 1.1.1.204) the primary gene product of XOR and as xanthine oxidase (XO) (1.1.3.22), a product of
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post-translational modifications of XDH. XDH favors the cofactor NAD+ as its primary electron acceptor, while XO is unable to bind to NAD+ and uses O2 as its electron acceptor. Through both forms, but particularly through catalysis by the XO form, numerous reactive oxygen species (ROS) are synthesized. The ability of XOR to rapidly convert from XDH into XO under the effect of the same cytokines that stimulate XOR expression in response to tissue damage makes XOR an ideal component of fast innate immune response (Vorbach et al., 2003; Martin et al., 2004). About 20 million people in America are infected with T. cruzi and 50,000 deaths per year are associated with the infection (World Health Organization, 2002). Our current study revealed that serum obtained from patients diagnosed with Chagas’ disease displays an inhibitory effect on the growth of epimastigotes of the T. cruzi RA strain when added to the growth medium. This activity was not present in the serum of healthy individuals, nor was it due to antibodies against the parasite or to the complement system. In this paper we characterize hydrogen peroxide present in the sera of chagasic patients, with or without cardiac manifestation, as being responsible for the inhibition of growth of the epimastigotes. The increase of hydrogen peroxide to antiproliferative levels might result from an increase in XO activity in the serum of patients infected with T. cruzi.
T. cruzi epimastigotes were grown at 28 ◦ C in liver infusion/tryptosa (LIT) medium containing the heat-inactivated test serum sample or fetal bovine serum (FBS) for the periods indicated in Section 3. During the incubation period the number of mobile epimastigotes was determined daily in a Neubauer chamber. Mobile parasites were considered viable while sluggish, immobile parasites were excluded. The parasites were counted in triplicate. Parasite viability was confirmed by Trypan blue assay (Freshney, 1994). 2.4. Serum heat inactivation All the assayed sera were placed in a 56 ◦ C water bath for 30 min in order to destroy heat labile complement proteins. 2.5. Antibody depletion
2. Materials and methods
For the removal of specific antibodies, serum samples were adsorbed on sensitized with T. cruzi surface antigens erythrocytes (Wiener Lab). Each patient serum was added to the erythrocyte pellet, then mixed and incubated at room temperature for 18 h. After incubation, the mixture was cleared by centrifugation. The procedure was repeated until the ELISA test for T. cruzi specific antibodies became negative. Experiments designed to test the antiproliferative effect of immunoglobulin depleted sera used the supernatant.
2.1. Serum samples
2.6. Serum dialysis
Serum samples were collected from adult patients diagnosed with Chagas’ disease (chagasic sera) and from adult individuals never diagnosed with Chagas’ disease (control human sera), at the Parasitology Laboratory of “José María Ramos Mejía Hospital”, Buenos Aires. These individuals agreed voluntarily to participate in the study by signing an Informed Consent Form. According to the clinical manifestations, the chagasic sera were classified as:
A 3 ml serum sample was placed in a semipermeable nitrocellulose membrane dialysis tube, molecular mass cut off: 6000–8000 and dialyzed with stirring against 15 ml LIT medium at 4 ◦ C for 12 h. Low molecular weight molecules passed through the dialysis membrane while proteins were retained in the tube. In order to asses the antiproliferative action of the low molecular weight fraction of the serum, the parasite pellet was resuspended in the LIT medium obtained after dialysis which contained 10% of the low molecular weight fraction of the serum. To study the effect of the high molecular weight fraction on the parasite growth the sealed tube was placed into fresh LIT medium and the dialysis was repeated twice. The high molecular weight fraction of the serum was added to the parasite growth medium at 10% concentration, as indicated in Section 3.
• Indeterminate chagasic sera (ICh), from patients with T. cruzi specific antibodies without clinical signs of cardiac abnormalities (85 individuals). • Cardiac chagasic sera (CCh), from patients with recognizable signs and symptoms of chagasic cardiomyopathy (15 individuals).
2.7. Xanthine oxidoreductase activity Non-chagasic sera were classified as: • Control sera (NCh), obtained from healthy individuals (70 individuals). • Non-chagasic cardiac (CNCh), from patients with cardiopathies not related to Chagas’ disease (12 individuals). All the sera, chagasic or non-chagasic, were obtained from individuals who had no other chronic inflammatory or autoimmune diseases at the time of the study. 2.2. Diagnosis of T. cruzi infection Patients were diagnosed positive via indirect hemagglutination (IHA) test (Wiener Lab.), indirect immunofluorescence assay (IIFA) and enzyme-linked immunoabsorbent assay, ELISA (Wiener Lab.).
Aliquots of sera were assayed in 50 mM sodium carbonate buffer containing 2.4 mM EDTA, 20 M cytochrome c and 2.4 mM xanthine. The mixtures were incubated at 37 ◦ C for 30 min and 10% trichloroacetic acid was added to each sample in order to stop the reaction. Cytochrome c reduction was monitored at 540 nm using a matched xanthine free negative control sample as blank. The specificity of the detection method was verified using allopurinol, a XOR specific inhibitor. To asses both total XOR (XO plus XDH) and XO activity, the reaction was performed respectively with and without NAD+ . In the presence of NAD+ , NADH is formed by XDH instead of H2 O2 plus O2 − , and ROS generation elicited by XOR decreased. As a result, the addition of NAD+ to the reaction mixture diminished cytochrome c reduction, and the remaining XOR activity is only due to XO (Muranjan et al., 1997). 2.8. Lipid peroxidation and superoxide dismutase activity
2.3. Organisms and media T. cruzi epimastigotes, RA strain were kindly provided by Dr. Estela Lammel and Dr. Elvira Isola from the Department of Parasitology, School of Medicine, Buenos Aires University.
The epimastigotes grown in the LIT medium were exposed to each tested serum for 24 h. The cells were then pelleted, homogenized and analyzed for lipid peroxides and superoxide dismutase (SOD) activity.
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The extent of lipid peroxidation was determined as the amount of the thiobarbituric acid-reactives substances (TBARS) in terms of malondialdehyde (MDA). Samples (0.2–0.5 mg protein) were heated with 1% (v/v) thiobarbituric acid at 100 ◦ C for 10 min. After cooling the absorbance was read at 532 nm and the concentration of TBARS calculated, based on a ε value of 150,000 M−1 cm−1 (Pompella et al., 1987). To determine SOD activity, the xanthine–xanthine oxidase system was used to generate O2 •− and the reduction of cytochrome c by O2 •− was monitored at 540 nm (Flohe and Otting, 1984). The inhibition of this reduction when SOD containing preparation was added, was used as an indicator of SOD activity. The reaction mixture contained 50 mM potassium phosphate buffer, pH 7.8, 0.1 mM EDTA, 50 M xanthine, 20 M cytochrome c, xanthine oxidase (0.2 U/ml) and 30 l of the homogenate. One unit of SOD activity is defined as the amount of enzyme that inhibits the rate of cytochrome c reduction by 50%.
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Fig. 1. Effect of whole and specific immunoglobulin free chagasic sera on growth of epimastigotes of Trypanosoma cruzi. Parasites were grown for 24 h in the presence of 10% (v/v) of the respective sera. ICh indetermined chagasic sera (n = 40), NCh nonchagasic sera (n = 30). The experiment was repeated twice. Results are expressed as mean ± S.D.
2.9. Protein content Was determined by Bradford method with crystalline bovine serum albumin as reference standard (Bradford, 1976). 2.10. Statistical analyses Data sets were compared by two-tailed Student’s t-test with two samples of unequal variance. Data sets were considered to be significantly different for p < 0.01.
with chagasic serum for 72 h stayed immobile and were unable to replicate when they were resuspended in fresh medium (data not shown). The antiproliferative action of chagasic sera increased in a concentration dependent manner. It was first observed at concentrations higher than 10%. At 20% the chagasic sera not only did not support parasite growth, but even killed the epimastigotes.
2.11. Chemicals Xanthine, xanthine oxidase, N-ethylmaleimide (NEM), cytochrome c, NAD+ , allopurinol, thiobarbituric acid and catalase were purchased from SIGMA Chemical Co. St. Louis, MO/USA. Fetal bovine serum (FBS) was from Gibco. All reagents were of analytical grade. 3. Results 3.1. Serum from chagasic patients display an antiproliferative effect on T. cruzi epimastigotes Serum from patients who had been diagnosed with Chagas disease, with (chronic) or without (indeterminate) cardiac manifestations, in which the complement system was inactivated by heat, presented an antiproliferative effect on T. cruzi epimastigotes RA strain. This effect was observed with whole serum as well as with serum previously depleted of the specific immunoglobulin fraction (Fig. 1). In contrast, this antiproliferative activity was absent in samples obtained from uninfected individuals with or without myocardiopathy (Fig. 2A and B), whether the sera were or were not depleted of immunoglobulins (Fig. 1). 3.2. The antiproliferative effect of chagasic sera on T. cruzi epimastigotes is time and concentration dependent The effect of 10% chagasic sera on the growth of the parasites was not immediate; a 14 h lag phase preceded the initiation of the antiproliferative action which increased after this point, in a time dependent manner: at 16 h, growth was 82%, at 24 h 70%, at 48 h 43% and at 72 h 16% as compared to the control groups grown in the presence of human sera from healthy individuals (Fig. 2A and B). Moreover, T. cruzi that had been incubated with chagasic sera for 16–48 h retained both their capacity to replicate when they were transferred to fresh medium and their growth characteristics. On the other hand, those trypanosomes that had been incubated
Fig. 2. Effect of chagasic and non-chagasic sera on growth of epimastigotes of T. cruzi. The parasites were incubated with 10% (v/v) human sera or FBS for the indicated time. At these times parasites were counted in a Neubauer chamber as indicated in Section 2. A: time in days; B: time in hours. FBS: fetal bovine serum; ICh: indetermined chagasic sera; NCh: non-chagasic sera; CCh: cardiac chagasic sera; CNCh: cardiac non-chagasic sera. The experiment was repeated twice. Results are expressed as mean ± S.D. (n = 9 for each serum).
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Fig. 4. Effect of dialysis on the antiproliferative action of chagasic serum. Sera were dialyzed as indicated in Section 2 and 10% (v/v) of high molecular weight fraction (H) or low molecular weight fraction (L) as well as complete sera (Co) were added to the growth medium for 24 h. FBS: fetal bovine sera; ICh: indetermined chagasic sera; NCh: non-chagasic sera. The experiment was repeated twice. Results are expressed as mean ± S.D. (n = 6 for each serum). p < 0.01 compared to the respective NCh and FBS.
Fig. 3. Effect of serum concentration on growth of epimastigotes of T. cruzi. Parasites were exposed for 24 h to the corresponding sera as described in Section 2. FBS: fetal bovine sera; ICh: indetermined chagasic sera; NCh: non-chagasic sera. The experiment was repeated twice. Results are expressed as mean ± S.D. (n = 10 for each serum).
3.4. XO activity is increased in the sera of chagasic patients
In contrast, parasite growth increased with higher concentrations of FBS or with normal human sera, reaching a maximum at 20% (Fig. 3).
The results presented in Table 1 showed that in sera from chagasic patients XOR activity was about twice as high as that found in both uninfected human sera and FBS. When the determination of XOR activity was performed in the presence of NAD+ , the remaining activity due to the XO form was 65% for chagasic sera, 37% for FBS and 42% for normal human sera, thus indicating that in the chagasic sera, XOR is predominantly present as XO (Table 1). Consequently, XO activity in chagasic sera proved to be three to four times higher than that seen for human sera or FBS respectively, instead of only twice as indicated for XOR.
3.3. A non-dialyzable, high molecular weight component is responsible for the antiproliferative effect of chagasic sera In order to characterize the nature of the antiproliferative serum component, sera were dialyzed against the growth medium as indicated in Section 2. Antiproliferative action was assayed in the presence of both the non-dialyzed, high molecular weight fraction and the dialyzed, low molecular weight one. Results showed that the antiproliferative effect of the high molecular weight fraction of chagasic sera was similar to that of the whole chagasic sera, while the low molecular weight fraction showed no antiproliferative action. No difference was observed between both high and low molecular weight fractions either with FBS or with human sera from healthy individuals on parasite proliferation (Fig. 4). The low molecular weight fraction of both chagasic and control sera were unable to support parasite proliferation beyond 24 h (data not shown)
3.5. XO and H2 O2 are involved in the antiproliferative effect of sera from chagasic patients Conversion of XDH to XO leads to the use of O2 as the electron acceptor during purine oxidation and results in the generation of reactive oxygen intermediates such as H2 O2 . Allopurinol – a suicide inhibitor of XOR–, catalase – an enzyme which degrades H2 O2 – and N-ethylmaleimide (NEM) – an inhibitor of H2 O2 catabolism in
Table 1 XOR and catalase activities in sera of both chagasic and non-chagasic patients and superoxide dismutase activity in epimastigotes of Trypanosoma cruzi grown during 24 h in the presence of 10% (v/v) different sera. In XOR assays, 80 M allopurinol or 0.5 mM NAD+ were added. XOR in sera (arbitrary units/ml)
Catalase in sera (mU/ml)
SOD in epimastigotes (U/mg prot)
FBS
−NAD+ 1.79 ± 0.29 +NAD+ 0.67 ± 0.17 +Allopurinol 0.73 ± 0.19
nd
0.99 ± 0.09
ICh
−NAD+ 4.20 ± 0.47* +NAD+ 2.79 ± 0.21* +Allopurinol 1.7 ± 0.15
84 ± 10
0.46 ± 0.12#
CCh
−NAD+ 5.30 ± 0.68* +NAD+ 1.80 ± 0.41*
nd
NCh
−NAD+ 2.20 ± 0.17 +NAD+ 1.01 ± 0.10 +Allopurinol 0.88 ± 0.19
94 ± 19
CNCh
−NAD+ 1.75 ± 0.49 +NAD+ 0.62 ± 0.19
nd
0.85 ± 0.17
Enzyme activities were assayed as described in Section 2. Results are expressed as mean ± S.D. (n = 15 for xanthine oxidase, n = 10 for catalase, n = 5 for SOD). nd: not determined. * p < 0.01 compared to non-chagasic sera (FBS, NCh, CNCh). # p < 0.01 compared to non-chagasic sera (FBS, NCh).
S.M. Hernandez et al. / Acta Tropica 109 (2009) 219–225
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Fig. 5. Effect of allopurinol, catalase and NEM on the growth of T. cruzi parasites exposed to different sera. 80 M allopurinol, 10 U/ml catalase, 200 M NEM and 10% (v/v) sera were added for 24 h. FBS: fetal bovine sera; ICh: indetermined chagasic sera; NCh: non-chagasic sera. Results are expressed as mean ± S.D. (n = 10 for each serum). p < 0.01 compared to the corresponding NCh and FBS. p < 0.01 compared to none ICh. None, allopurinol, catalase, allopurinol + catalase, NEM.
epimastigotes–, were used in order to support the role of XO and its product H2 O2 in the antiproliferative action of serum from chagasic patients. The addition of allopurinol (80 M) to the reaction mixture produced a 60% decrease in XOR activity (Table 1). Furthermore allopurinol increased parasite proliferation 65% when added to the growth medium containing chagasic sera. These effects of the XOR specific inhibitor indicate the participation of the enzyme in the antiproliferative action of chagasic sera. The addition of catalase (10 U/ml) to the medium containing chagasic sera resulted in a 100% increase in parasite proliferation. This result suggests the involvement of H2 O2 in the antiproliferative effect of chagasic sera. Allopurinol and catalase did not show additive effect when added jointly as growth rate did not differ from the values observed with catalase alone (Fig. 5) indicating that XO was not the only source of H2 O2 . When allopurinol or catalase were added to growth medium containing FBS or human sera from non-infected individuals, no significant difference in growth as compared with controls, was observed (Fig. 5). NEM concentration was selected in order to avoid any damage on the growth of the parasites grown in a medium supplemented with FBS. This selected NEM concentration did not affect parasite proliferation in the presence of human non-chagasic sera. The addition of NEM, however, resulted in a 50% elevation of the antiproliferative effect of chagasic sera (Fig. 5) which is in agreement with an increase in H2 O2 production. Serum catalase activity was measured in order to asses whether the XO mediated increase in H2 O2 production was affected by changes in the activity of this enzyme. Table 1 shows that catalase activity did not differ significantly between human sera from chagasic and non-chagasic individuals.
Determination of SOD activity in the parasites treated with the different sera showed that SOD activity was almost half as low in parasites grown in the presence of chagasic sera than in those incubated with FBS or normal human sera (Table 1) indicating a decrease in the defense mechanism against ROS. In conclusion these results suggest that the antiproliferative effect of chagasic sera could be due to oxidative stress. 4. Discussion It has been known since 1912 (Laveran and Mesnil, 1912) that normal human serum possesses innate protection against infection by the pathogen T. brucei brucei, but not against the agent of human
3.6. Sera from chagasic patients increase lipoperoxidation of T. cruzi epimastigotes membranes and decrease parasite SOD activity To determine whether an oxidative damage was involved in the trypanolytic action of the sera from chagasic patients, lipid peroxidation of polyunsaturated fatty acids of the parasite membranes was measured. Fig. 6 shows that there was a dose dependent increase in lipoperoxidation of the membranes of parasites treated with chagasic sera (Fig. 6). In contrast, an increase in FBS in the culture medium exerted a protective role against lipoperoxidation, while, at the highest assayed doses, normal human sera did not affect basal lipoperoxidation (Fig. 6).
Fig. 6. Effects of different sera on the lipoperoxidation of T. cruzi epimastigote membranes. Parasites were grown for 24 h in the presence of 10% (v/v) or 15% (v/v) of each serum. After this time parasites were harvested and homogenized to determine MDA levels. FBS: fetal bovine sera; ICh: indetermined chagasic sera; NCh: non-chagasic sera; CCh: chagasic patients with cardiopathy. Results are expressed as mean ± S.D. (n = 15 for each serum). p < 0.01 compared to FBS or NCh.
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African sleeping sickness, T. brucei rhodesiense. The observed protection is a result of non-immune killing factors present in human sera, known as trypanosome lytic factors (TLF). Two TLFs have been identified in human sera as circulating lipoprotein complexes, a subset of high density lipoprotein (HDL), which contain – in addition to apoA-1–, haptoglobin related protein, apolipoprotein L-1, and immunoglobulin M (Molina Portela et al., 2000; Raper et al., 2001; Vanhamme and Pays, 2004). Humans are susceptible to infection by T. cruzi. Nevertheless, human sera exhibit a trypanolytic action due to thermolabile factors one of which is related to the complement system and inhibits trypanosome epimastigote growth in vitro. The other is related to anti-alpha-galactosyl-antibodies which recognize the F2/3 antigenic fraction (Gazzinelli and Pereira, 1994; Almeida et al., 1994; Altcheh et al., 2003). The trypanolytic activity of normal human sera is abolished by heat inactivation at 56 ◦ C for 30 min. The data presented in this study show that sera from chagasic patients depleted of the complement system, displayed an antiproliferative action on T. cruzi epimastigotes, RA strain, when added to the growth medium, thereby indicating the presence of factors affecting the growth of the parasites other than those of the complement system. The antiproliferative effect was observed with sera of chagasic patients containing or depleted of specific antibodies, obtained from individuals without clinical manifestations or with cardiomyopathy. The antiproliferative effect was absent from the sera of uninfected healthy individuals and from uninfected patients with cardiovascular disease. Hence, restrained trypanosome growth may be due to the presence of growth inhibitory serum components other than specific antibodies or the complement system, induced by the infection and unrelated to the cardiomyopathy of the patient. The antiproliferative activity of sera from chagasic patients presented both concentration and time dependent behavior. A lytic effect was observed when the parasites were incubated with chagasic sera for 72 h at any of the assayed concentrations or at least for 24 h with 20% serum. Treatment induced parasite immobility, followed by complete growth arrest, loss of replication capacity upon transfer to fresh medium and eventual death. On the other hand, with serum doses below 20% or incubation times lower than 72 h, a trypanostatic action on the epimastigotes was observed; although the parasites stopped growing, they retained both their mobility and their capacity to proliferate when resuspended in a fresh medium. Neither trypanolytic nor trypanostatic effects were observed with FBS or with sera from uninfected individuals, suggesting that these effects were due to materials present in human serum produced post-infection. Sera were dialyzed as a first step towards the determination of the nature of the trypanolytic or trypanostatic serum component. The results obtained indicated that the antiproliferative material present in the chagasic serum was a non-dialyzable, high molecular weight substance. Taking into account that the antiproliferative effect was observed only in the sera of patients infected with T. cruzi and that XOR has been reported to participate in the systemic antimicrobial response of the innate immune system (Vorbach et al., 2003), we studied the activities of this enzyme in the different sera. Healthy individuals have low levels of circulating XOR (Martin et al., 2004). An increase in the enzyme activity in response to a range of diseases like endothelial dysfunction, hypertension, heart failure, and diabetes has been reported (Martin et al., 2004; Desco et al., 2002; Berry and Hare, 2004). In this study we report for the first time that the levels of XOR and XO increased considerably in all sera from both cardiac and indeterminate chagasic patients, presumably as a response to T. cruzi invasion. The infection elicits IFN-␥ production by natural killer cells and activates phagocytic cells, thus increasing hydrogen peroxide, nitric
oxide and TNF-␣ production (Cardoni, 1997; Samudio et al., 1998) all of which are essential for controlling acute parasitemia. XOR expression is stimulated by IFN-␥, IFN-␣ TNF-␣, IL-1 and IL-3. Some of these factors also initiate the conversion from XDH to XO, thus increasing ROS generation (Berry and Hare, 2004). It was reported that the Th1 pattern of immune response predominates during the entire course of the Chagas disease, including the chronic stage (Nabors and Tarleton, 1991; Cardoni et al., 1999; Antunez and Cardoni, 2000). This is consistent with our observations that both XOR and XO activities increased significantly in the serum of patients in the chronic phase of the disease. Although both laboratory and clinical investigations have reported an increase in the levels of endothelial bound XOR activity in a number of cardiovascular diseases (Berry and Hare, 2004), we observed that circulating XOR and XO activities in non-chagasic patients with dilated cardiopathy did not differ significantly from those of the control group (Table 1). The lack of increase in serum XOR activities in non-chagasic patients with dilated cardiopathy may be related to a different etiology of the disease, not involving oxidative stress. Lipid peroxidation is a major biomarker of ROS-generated oxidative damage. Increased lipid peroxidation of epimastigote membranes elicited by chagasic sera indicated the involvement of oxidative stress in the trypanolytic effect. The fact that sera of healthy individuals did not display an increase in XO activity or in ROS production, as evidenced by the maintenance of lipoperoxidation of parasite membranes at basal values, underlines the importance of the infection in triggering these processes. The antiproliferative effect of chagasic sera was decreased by both allopurinol – an inhibitor of XO–, and catalase – an enzyme that degrades hydrogen peroxide–. On the contrary, the addition of NEM, which by inhibiting hydrogen peroxide catabolism by the epimastigotes increases the levels of hydrogen peroxide, enhanced the antiproliferative activity. Taken together these results and our previous observation that the addition of hydrogen peroxide to the growth medium in the presence of NEM resulted in the death of the epimastigotes of T. cruzi (Hernandez et al., 2006), we conclude that the observed effects on parasite growth and viability are likely due to an increase in the concentration of hydrogen peroxide in the serum of the chagasic patients as a result of increase in XO activity. Moreover, this proposal is reinforced by the fact that SOD activity was much lower in parasites grown in the presence of chagasic sera since our previous studies and that of other authors demonstrated that the addition of hydrogen peroxide to the growth medium actually reduced the Fe/SOD activity in the epimastigote (Hernandez et al., 2006; Ismail et al., 1997). A consistent decline in Mn/SOD activity, the major oxygen radical scavenger in the mitochondrial matrix, during progression of infection and disease in chagasic myocardium has also been reported (Wen et al., 2004). Hydrogen peroxide can be generated by different types of oxidases. The fact that allopurinol, a specific inhibitor of XO, decreased the hydrogen peroxide dependent antiproliferative effect of chagasic sera, suggested that XO plays an important role in ROS generation in the sera of chagasic patients. Nevertheless we cannot discard the possible existence in the plasma of chagasic patients of other peroxide generating systems such as SOD and polyamineoxidases. In Cape Buffalo the natural resistance to T. brucei parasitemia has been attributed to hydrogen peroxide derived from serum XO. The increase in hydrogen peroxide correlates with a five to eightfold decline in blood catalase with no change in XO activity (Wang et al., 1999; Black et al., 2001). In contrast to the results obtained with Cape Buffalo, in the serum of chagasic patients at the chronic phase of the disease, we observed an increase in oxidative stress resulting from an increase in XO activity rather than from a decrease in catalase activity.
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In humans, sterilizing immunity does not appear to exist in T. cruzi infection. The XO dependent-ROS production triggered by the infection does not seem to have an antimicrobial role. Nevertheless, the continuous exposition to ROS thus generated, might contribute to the development of chagasic cardiomyopathy (Zacks et al., 2005). In conclusion, the data discussed so far provides evidence to support the idea that the antiproliferative activity observed in sera from chagasic patients may be due, among other factors, to a direct effect of hydrogen peroxide on the epimastigotes of T. cruzi. One of the greatest concerns in Chagas’ disease is the absence of reliable methods for the evaluation of chemotherapy efficacy in treated patients. The differences between sera of both infected and uninfected individuals shown in this study, may potentially be used to develop a reliable and safe test to follow both the evolution of the Chagas disease and the response to pharmacological treatment in patients. Acknowledgements Authors are very thankful to Dr. Elvira D. Isola and Dr. Estela Lammel from the Department of Microbiology, Parasitology and Inmunology (School of Medicine, University of Buenos Aires) for generously providing T. cruzi epimastigotes, to Dr. Alicia Fuchs for critical review and to Marianne Revah, Carolina Soleil and Melina Meneguin for language revision of the manuscript. This research was supported by a grant (PICTO 31428) from the Agencia Nacional de promoción Científica y Tecnológica and by the Universidad Abierta Interamericana. References Almeida, I.C., Ferguson, M.A.J., Schenkman, S., Travassos, L.R., 1994. Lytic anti␣-galactosil antibodies from patients with chronic Chagas disease recognize novel O-linked oligosaccharides on mucin-like glycosyl-phosphatidylinositolanchored glyproteins of Trypanosoma cruzi. Biochem. J. 304, 793–802. Altcheh, J., Corral, R., Biancardi, M., Freilij, H., 2003. Anticuerpos anti-F2/3 como ˜ con infección congénita por Trypanosoma cruzi. marcador de curación en ninos Medicina 63, 37–44. Antunez, M., Cardoni, R., 2000. IL-12 and INF ␥ production, and NK cell activity, in acute and chronic experimental Trypanosoma cruzi infections. Immunol. Lett. 71, 103–109. Berry, C.E., Hare, J.M., 2004. Xantine oxidoreductase and cardiovascular disease. Mechanism and physiological implications. J. Physiol. 555, 589–606. Black, S., Sicard, E., Murphy, N., Nolan, D., 2001. Innate and acquired control of trypanosome parasitaemia in Cape Buffalo. Int. J. Parasitol. 31, 562–565. Bradford, M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 7, 248–254. Cardoni, R., 1997. La respuesta inflamatoria en la infección aguda con Trypanosoma cruzi. Medicina 57, 227–234. Cardoni, R., Antunez, M., Abrami, A., 1999. Respuesta TH1 en la infección experimental con Trypanosoma cruzi. Medicina 59, 84–90. Cross, G.A.M., 1990. Cellular and genetic aspect of antigenic variation in Trypanosome. Annu. Rev. Immunol. 8, 83–110.
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Desco, M.C., Asensi, M., Marquez, R., Martinez-Valls, J., Vento, M., Pallardo, F.V., Sastre, ˜ J., 2002. Xanthine oxidase is involved in free radical production in type J., Vina, 1 diabetes. Protection by allopurinol. Diabetes 5, 1118–1128. Drain, J., Bishop, J., Hajduk, S., 2001. Haptoglobine-related protein mediates trypanosome lytic factor binding to trypanosomes. J. Biol. Chem. 32, 30254–30260. Flohe, L., Otting, F., 1984. Superoxide dismutase assays. Methods Enzymol. 105, 93–104. Freshney, R.L., 1994. The Culture of Animal Cells: A Manual of Basic Technique, 3rd ed. Wiley–Liss, NY, USA. Gazzinelli, R.T., Pereira, M.E., 1994. Direct lysis of Trypanosoma cruzi: a novel effector mechanism of protection mediated by human anti-gal antibodies. J. Eukaryot. Microbiol. 41, 47–57. Hernandez, S.M., Sanchez, M.S., Schwarcz de Tarlovsky, M.N., 2006. Polyamine as a defense mechanism against lipoperoxidation in Trypanosoma cruzi. Acta Trop. 98, 94–102. Ismail, S.O., Paramchuk, W., Skeiky, Y., Reed, S., Bhatia, A., Gedamu, L., 1997. Molecular cloning and characterization of two iron superoxide dismutase cDNAs from Trypanosoma cruzi. Mol. Biochem. Parasitol. 86, 187–197. Laveran, A., Mesnil, F., 1912. In: Masson, S. (Ed.), Trypanosomes et trypanosomiases. Machado, C., Augusto-Pinto, L., McCulloch, R., Teixeira, S., 2006. DNA metabolism and genetic diversity in Trypanosomes. Mut. Res. 612, 40–57. Martin, H., Hancock, J., Salisbury, V., Harrison, R., 2004. Role of xanthine oxidoreductase as an antimicrobial agent. Infect. Immun. 72, 4933–4939. Molina Portela, M., Raper, J., Tomlinsom, S., 2000. An investigation into the mechanism of Trypanosome lysis by human factors. Mol. Biochem. Parasitol. 110, 273–282. Muranjan, M., Wan, Q., Li, Y., Hamilton, E., Otieno-Omondi, F.P., Wang, J., Van Praagh, A., Grootenhuis, J.G., Black, S.J., 1997. The trypanocidal Cape Buffalo serum protein is xanthine oxidase. Infect. Immun. 65, 6806–6814. Nabors, G., Tarleton, R., 1991. Differential control of IFN-␥ and IL-2 production during Trypanosoma cruzi infection. J. Immunol. 146, 3591–3598. Pays, E., Vanhollebeke, B., Vanhamme, L., Paturiaux-Hanocq, F., Nolan, D., PerezMorga, D., 2006. The trypanolytic factor of human serum. Nat. Rev. Microbiol. 4, 477–486. Pompella, A., Maellaro, E., Casini, A.F., Ferrali, M., Ciccoli, L., Comporti, M., 1987. Measurement of lipid peroxidation in vivo: a comparison of different procedures. Lipids 22, 206–211. Raper, J., Fung, R., Ghiso, J., Nussenweig, V., Tomlinson, S., 1999. Characterization of novel trypanosome lytic factor from human serum. Infect. Immun. 67, 1910–1916. Raper, J., Portela, M.P., Lugli, E., Freverd, U., Tomlinson, S., 2001. Trypanosome lytic factors: novel mediators of human innate immunity. Curr. Opin. Microbiol. 4, 402–408. Samudio, M., Montenegro-James, S., Cabral, M., Martinez, J., Rojas de Arias, A., James, M., 1998. Cytokine responses in T. cruzi infected children in Paraguay. Am. J. Trop. Med. Hyg. 58, 119–121. Vanhamme, L., Pays, E., 2004. The trypanosome lytic factor of human serum and the molecular basis of sleeping sickness. Int. J. Parasitol. 34, 887–898. Vorbach, C., Harrison, R., Capecchi, M.R., 2003. Xanthine oxidoreductase is central to the evolution and function of the innate immune system. Trends Immunol. 24, 512–517. Wang, Q., Murphy, N., Black, S.J., 1999. Infection-associated decline of Cape Buffalo blood catalase augments serum trypanocidal activity. Infect. Immun. 6, 2797–2803. Wen, J.J., Vyatkina, G., Garg, N., 2004. Oxidative damage during chagasic cardiomyopathy development: role of mitochondrial oxidant release and inefficient antioxidant defense. Free Rad. Biol. Med. 37, 1821–1833. World Health Organization, 2002. Control of Chagas disease: Second report of the World Health Organization expert committee Strategic Direction for Research Geneva, Switzerland, pp. 1–159. Zacks, M., Wen, J., Vyatkina, G., Bhatia, V., Garg, N., 2005. An overview of chagasic cardiomyopathy. Pathogenic importance of oxidative stress. Anais da Acad. Bras. de Ciencias 77, 695–714.
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Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Antiproliferative effect of sera from chagasic patients on Trypanosoma cruzi epimastigotes. Involvement of xanthine oxidase Susana M. Hernandez a , Rodolfo A. Kolliker-Frers b , Marcela S. Sanchez c , Gabriela Razzitte b , Rodny D. Britos a , Maria E. Fuentes a , Martha N. Schwarcz de Tarlovsky a,∗ a b c
School of Medicine, CAECIS, Universidad Abierta Interamericana, Montes de Oca 745, 1270 Buenos Aires, Argentina Laboratory of Parasitology, Jose Maria Ramos Mejia Hospital, Urquiza 609, 1211 Buenos Aires, Argentina Ciclo Básico Común, Universidad de Buenos Aires, Paraguay 2155, 1121 Buenos Aires, Argentina
a r t i c l e
i n f o
Article history: Received 20 August 2008 Received in revised form 10 November 2008 Accepted 14 November 2008 Available online 25 November 2008 Keywords: Trypanosoma cruzi Chagas disease Xanthine oxidase Reactive oxygen species Hydrogen peroxide
a b s t r a c t Serum from asymptomatic or symptomatic (with cardiovascular manifestations) chagasic patients depleted of the complement system displayed an antiproliferative effect on Trypanosoma cruzi epimastigotes, RA strain, when added to the growth medium. This effect was also observed when patient’s serum was depleted of specific antibodies. The antiproliferative effect was both time and concentration dependent. It was confined to the nondialyzable, high molecular weight, fraction of the serum. This effect was abrogated by allopurinol and catalase, and enhanced by N-ethylmaleimide. Xanthine oxidoreductase and xanthine oxidase activities were increased in the chagasic sera, while catalase activity remained unchanged. Parasites exposed to chagasic sera showed a decrease in Fe/superoxide dismutase activity as well as an increase in membrane lipoperoxidation. Our data provides evidence to support the idea that the antiproliferative activity observed in sera from chagasic patients may be due, at least partially, to a direct effect of hydrogen peroxide on the epimastigotes of T. cruzi. The increase of hydrogen peroxide to antiproliferative levels might result from an increase in xanthine oxidase activity in the serum of patients infected with the parasite. © 2009 Published by Elsevier B.V.
1. Introduction The trypanosomiases are a group of diseases affecting humans and livestock in Africa, Asia and South America. These diseases are caused by the infection of blood-dwelling protozoan parasites called Trypanosoma. Trypanosoma brucei subspecies T. brucei gambiense and T. brucei rhodesiense are the etiologic agents of sleeping sickness (African trypanosomiasis) while T. cruzi is the causative agent of Chagas disease (American Trypanosomiasis). Both parasites developed the genetic variability needed for survival in their host, by using different strategies. T. brucei changes periodically the expression of a group of variant surface glycoproteins (Cross, 1990; Vanhamme and Pays, 2004), whereas T. cruzi counts on the extreme heterogeneity of their population. The source of T. cruzi antigenic diversity may arise from metabolic changes in the mismatch repair pathway (Machado et al., 2006). Nevertheless human blood, unlike the blood of other mammals, has an efficient trypanolytic activity against Trypanosoma brucei brucei. This is due to the presence of non-immune serum factors that lyse the invading trypanosome. One lytic factor of human serum
∗ Corresponding author. Tel.: +54 11 4301 5240; fax: +54 11 4301 5240x107. E-mail address: [email protected] (M.N. Schwarcz de Tarlovsky). 0001-706X/$ – see front matter © 2009 Published by Elsevier B.V. doi:10.1016/j.actatropica.2008.11.013
against T. brucei brucei was characterized as a subset of high density lipoproteins (HDL) which invariably include haptoglobin related protein and apolipoprotein L1 (Raper et al., 1999; Drain et al., 2001; Pays et al., 2006). On the other hand, it has been observed that only Cape buffalo, among a wide variety of domestic animals, is resistant to infection by African trypanosomes. This resistance is due to accumulation of a trypanocydal concentration of hydrogen peroxide in the buffalo serum, during catabolism of xanthine by xanthine oxidase as a consequence of the infection (Muranjan et al., 1997; Black et al., 2001). Infection, as well as various forms of tissue damage, induces inflammatory reactions as an important part of innate immunity. The inflammatory reaction results in the expression of a number of cytokines. Among these cytokines, gamma-interferon (IFN␥), alpha-interferon (IFN-␣), tumor necrosis factor-alpha (TNF-␣), interleukin-1 (IL-1) and interleukin-3 (IL-3) stimulate xanthine oxidoreductase (XOR) expression (Berry and Hare, 2004). XOR is a housekeeping enzyme with a role in purine catabolism, detoxification and the regulation of the cellular redox potential. The XOR enzyme is a homodimer composed of catalytically independent subunits with an approximate molecular mass of 150 kDa each. It exists in two inter-convertible enzymatic forms, as xanthine dehydrogenase (XDH) (E 1.1.1.204) the primary gene product of XOR and as xanthine oxidase (XO) (1.1.3.22), a product of
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post-translational modifications of XDH. XDH favors the cofactor NAD+ as its primary electron acceptor, while XO is unable to bind to NAD+ and uses O2 as its electron acceptor. Through both forms, but particularly through catalysis by the XO form, numerous reactive oxygen species (ROS) are synthesized. The ability of XOR to rapidly convert from XDH into XO under the effect of the same cytokines that stimulate XOR expression in response to tissue damage makes XOR an ideal component of fast innate immune response (Vorbach et al., 2003; Martin et al., 2004). About 20 million people in America are infected with T. cruzi and 50,000 deaths per year are associated with the infection (World Health Organization, 2002). Our current study revealed that serum obtained from patients diagnosed with Chagas’ disease displays an inhibitory effect on the growth of epimastigotes of the T. cruzi RA strain when added to the growth medium. This activity was not present in the serum of healthy individuals, nor was it due to antibodies against the parasite or to the complement system. In this paper we characterize hydrogen peroxide present in the sera of chagasic patients, with or without cardiac manifestation, as being responsible for the inhibition of growth of the epimastigotes. The increase of hydrogen peroxide to antiproliferative levels might result from an increase in XO activity in the serum of patients infected with T. cruzi.
T. cruzi epimastigotes were grown at 28 ◦ C in liver infusion/tryptosa (LIT) medium containing the heat-inactivated test serum sample or fetal bovine serum (FBS) for the periods indicated in Section 3. During the incubation period the number of mobile epimastigotes was determined daily in a Neubauer chamber. Mobile parasites were considered viable while sluggish, immobile parasites were excluded. The parasites were counted in triplicate. Parasite viability was confirmed by Trypan blue assay (Freshney, 1994). 2.4. Serum heat inactivation All the assayed sera were placed in a 56 ◦ C water bath for 30 min in order to destroy heat labile complement proteins. 2.5. Antibody depletion
2. Materials and methods
For the removal of specific antibodies, serum samples were adsorbed on sensitized with T. cruzi surface antigens erythrocytes (Wiener Lab). Each patient serum was added to the erythrocyte pellet, then mixed and incubated at room temperature for 18 h. After incubation, the mixture was cleared by centrifugation. The procedure was repeated until the ELISA test for T. cruzi specific antibodies became negative. Experiments designed to test the antiproliferative effect of immunoglobulin depleted sera used the supernatant.
2.1. Serum samples
2.6. Serum dialysis
Serum samples were collected from adult patients diagnosed with Chagas’ disease (chagasic sera) and from adult individuals never diagnosed with Chagas’ disease (control human sera), at the Parasitology Laboratory of “José María Ramos Mejía Hospital”, Buenos Aires. These individuals agreed voluntarily to participate in the study by signing an Informed Consent Form. According to the clinical manifestations, the chagasic sera were classified as:
A 3 ml serum sample was placed in a semipermeable nitrocellulose membrane dialysis tube, molecular mass cut off: 6000–8000 and dialyzed with stirring against 15 ml LIT medium at 4 ◦ C for 12 h. Low molecular weight molecules passed through the dialysis membrane while proteins were retained in the tube. In order to asses the antiproliferative action of the low molecular weight fraction of the serum, the parasite pellet was resuspended in the LIT medium obtained after dialysis which contained 10% of the low molecular weight fraction of the serum. To study the effect of the high molecular weight fraction on the parasite growth the sealed tube was placed into fresh LIT medium and the dialysis was repeated twice. The high molecular weight fraction of the serum was added to the parasite growth medium at 10% concentration, as indicated in Section 3.
• Indeterminate chagasic sera (ICh), from patients with T. cruzi specific antibodies without clinical signs of cardiac abnormalities (85 individuals). • Cardiac chagasic sera (CCh), from patients with recognizable signs and symptoms of chagasic cardiomyopathy (15 individuals).
2.7. Xanthine oxidoreductase activity Non-chagasic sera were classified as: • Control sera (NCh), obtained from healthy individuals (70 individuals). • Non-chagasic cardiac (CNCh), from patients with cardiopathies not related to Chagas’ disease (12 individuals). All the sera, chagasic or non-chagasic, were obtained from individuals who had no other chronic inflammatory or autoimmune diseases at the time of the study. 2.2. Diagnosis of T. cruzi infection Patients were diagnosed positive via indirect hemagglutination (IHA) test (Wiener Lab.), indirect immunofluorescence assay (IIFA) and enzyme-linked immunoabsorbent assay, ELISA (Wiener Lab.).
Aliquots of sera were assayed in 50 mM sodium carbonate buffer containing 2.4 mM EDTA, 20 M cytochrome c and 2.4 mM xanthine. The mixtures were incubated at 37 ◦ C for 30 min and 10% trichloroacetic acid was added to each sample in order to stop the reaction. Cytochrome c reduction was monitored at 540 nm using a matched xanthine free negative control sample as blank. The specificity of the detection method was verified using allopurinol, a XOR specific inhibitor. To asses both total XOR (XO plus XDH) and XO activity, the reaction was performed respectively with and without NAD+ . In the presence of NAD+ , NADH is formed by XDH instead of H2 O2 plus O2 − , and ROS generation elicited by XOR decreased. As a result, the addition of NAD+ to the reaction mixture diminished cytochrome c reduction, and the remaining XOR activity is only due to XO (Muranjan et al., 1997). 2.8. Lipid peroxidation and superoxide dismutase activity
2.3. Organisms and media T. cruzi epimastigotes, RA strain were kindly provided by Dr. Estela Lammel and Dr. Elvira Isola from the Department of Parasitology, School of Medicine, Buenos Aires University.
The epimastigotes grown in the LIT medium were exposed to each tested serum for 24 h. The cells were then pelleted, homogenized and analyzed for lipid peroxides and superoxide dismutase (SOD) activity.
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The extent of lipid peroxidation was determined as the amount of the thiobarbituric acid-reactives substances (TBARS) in terms of malondialdehyde (MDA). Samples (0.2–0.5 mg protein) were heated with 1% (v/v) thiobarbituric acid at 100 ◦ C for 10 min. After cooling the absorbance was read at 532 nm and the concentration of TBARS calculated, based on a ε value of 150,000 M−1 cm−1 (Pompella et al., 1987). To determine SOD activity, the xanthine–xanthine oxidase system was used to generate O2 •− and the reduction of cytochrome c by O2 •− was monitored at 540 nm (Flohe and Otting, 1984). The inhibition of this reduction when SOD containing preparation was added, was used as an indicator of SOD activity. The reaction mixture contained 50 mM potassium phosphate buffer, pH 7.8, 0.1 mM EDTA, 50 M xanthine, 20 M cytochrome c, xanthine oxidase (0.2 U/ml) and 30 l of the homogenate. One unit of SOD activity is defined as the amount of enzyme that inhibits the rate of cytochrome c reduction by 50%.
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Fig. 1. Effect of whole and specific immunoglobulin free chagasic sera on growth of epimastigotes of Trypanosoma cruzi. Parasites were grown for 24 h in the presence of 10% (v/v) of the respective sera. ICh indetermined chagasic sera (n = 40), NCh nonchagasic sera (n = 30). The experiment was repeated twice. Results are expressed as mean ± S.D.
2.9. Protein content Was determined by Bradford method with crystalline bovine serum albumin as reference standard (Bradford, 1976). 2.10. Statistical analyses Data sets were compared by two-tailed Student’s t-test with two samples of unequal variance. Data sets were considered to be significantly different for p < 0.01.
with chagasic serum for 72 h stayed immobile and were unable to replicate when they were resuspended in fresh medium (data not shown). The antiproliferative action of chagasic sera increased in a concentration dependent manner. It was first observed at concentrations higher than 10%. At 20% the chagasic sera not only did not support parasite growth, but even killed the epimastigotes.
2.11. Chemicals Xanthine, xanthine oxidase, N-ethylmaleimide (NEM), cytochrome c, NAD+ , allopurinol, thiobarbituric acid and catalase were purchased from SIGMA Chemical Co. St. Louis, MO/USA. Fetal bovine serum (FBS) was from Gibco. All reagents were of analytical grade. 3. Results 3.1. Serum from chagasic patients display an antiproliferative effect on T. cruzi epimastigotes Serum from patients who had been diagnosed with Chagas disease, with (chronic) or without (indeterminate) cardiac manifestations, in which the complement system was inactivated by heat, presented an antiproliferative effect on T. cruzi epimastigotes RA strain. This effect was observed with whole serum as well as with serum previously depleted of the specific immunoglobulin fraction (Fig. 1). In contrast, this antiproliferative activity was absent in samples obtained from uninfected individuals with or without myocardiopathy (Fig. 2A and B), whether the sera were or were not depleted of immunoglobulins (Fig. 1). 3.2. The antiproliferative effect of chagasic sera on T. cruzi epimastigotes is time and concentration dependent The effect of 10% chagasic sera on the growth of the parasites was not immediate; a 14 h lag phase preceded the initiation of the antiproliferative action which increased after this point, in a time dependent manner: at 16 h, growth was 82%, at 24 h 70%, at 48 h 43% and at 72 h 16% as compared to the control groups grown in the presence of human sera from healthy individuals (Fig. 2A and B). Moreover, T. cruzi that had been incubated with chagasic sera for 16–48 h retained both their capacity to replicate when they were transferred to fresh medium and their growth characteristics. On the other hand, those trypanosomes that had been incubated
Fig. 2. Effect of chagasic and non-chagasic sera on growth of epimastigotes of T. cruzi. The parasites were incubated with 10% (v/v) human sera or FBS for the indicated time. At these times parasites were counted in a Neubauer chamber as indicated in Section 2. A: time in days; B: time in hours. FBS: fetal bovine serum; ICh: indetermined chagasic sera; NCh: non-chagasic sera; CCh: cardiac chagasic sera; CNCh: cardiac non-chagasic sera. The experiment was repeated twice. Results are expressed as mean ± S.D. (n = 9 for each serum).
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Fig. 4. Effect of dialysis on the antiproliferative action of chagasic serum. Sera were dialyzed as indicated in Section 2 and 10% (v/v) of high molecular weight fraction (H) or low molecular weight fraction (L) as well as complete sera (Co) were added to the growth medium for 24 h. FBS: fetal bovine sera; ICh: indetermined chagasic sera; NCh: non-chagasic sera. The experiment was repeated twice. Results are expressed as mean ± S.D. (n = 6 for each serum). p < 0.01 compared to the respective NCh and FBS.
Fig. 3. Effect of serum concentration on growth of epimastigotes of T. cruzi. Parasites were exposed for 24 h to the corresponding sera as described in Section 2. FBS: fetal bovine sera; ICh: indetermined chagasic sera; NCh: non-chagasic sera. The experiment was repeated twice. Results are expressed as mean ± S.D. (n = 10 for each serum).
3.4. XO activity is increased in the sera of chagasic patients
In contrast, parasite growth increased with higher concentrations of FBS or with normal human sera, reaching a maximum at 20% (Fig. 3).
The results presented in Table 1 showed that in sera from chagasic patients XOR activity was about twice as high as that found in both uninfected human sera and FBS. When the determination of XOR activity was performed in the presence of NAD+ , the remaining activity due to the XO form was 65% for chagasic sera, 37% for FBS and 42% for normal human sera, thus indicating that in the chagasic sera, XOR is predominantly present as XO (Table 1). Consequently, XO activity in chagasic sera proved to be three to four times higher than that seen for human sera or FBS respectively, instead of only twice as indicated for XOR.
3.3. A non-dialyzable, high molecular weight component is responsible for the antiproliferative effect of chagasic sera In order to characterize the nature of the antiproliferative serum component, sera were dialyzed against the growth medium as indicated in Section 2. Antiproliferative action was assayed in the presence of both the non-dialyzed, high molecular weight fraction and the dialyzed, low molecular weight one. Results showed that the antiproliferative effect of the high molecular weight fraction of chagasic sera was similar to that of the whole chagasic sera, while the low molecular weight fraction showed no antiproliferative action. No difference was observed between both high and low molecular weight fractions either with FBS or with human sera from healthy individuals on parasite proliferation (Fig. 4). The low molecular weight fraction of both chagasic and control sera were unable to support parasite proliferation beyond 24 h (data not shown)
3.5. XO and H2 O2 are involved in the antiproliferative effect of sera from chagasic patients Conversion of XDH to XO leads to the use of O2 as the electron acceptor during purine oxidation and results in the generation of reactive oxygen intermediates such as H2 O2 . Allopurinol – a suicide inhibitor of XOR–, catalase – an enzyme which degrades H2 O2 – and N-ethylmaleimide (NEM) – an inhibitor of H2 O2 catabolism in
Table 1 XOR and catalase activities in sera of both chagasic and non-chagasic patients and superoxide dismutase activity in epimastigotes of Trypanosoma cruzi grown during 24 h in the presence of 10% (v/v) different sera. In XOR assays, 80 M allopurinol or 0.5 mM NAD+ were added. XOR in sera (arbitrary units/ml)
Catalase in sera (mU/ml)
SOD in epimastigotes (U/mg prot)
FBS
−NAD+ 1.79 ± 0.29 +NAD+ 0.67 ± 0.17 +Allopurinol 0.73 ± 0.19
nd
0.99 ± 0.09
ICh
−NAD+ 4.20 ± 0.47* +NAD+ 2.79 ± 0.21* +Allopurinol 1.7 ± 0.15
84 ± 10
0.46 ± 0.12#
CCh
−NAD+ 5.30 ± 0.68* +NAD+ 1.80 ± 0.41*
nd
NCh
−NAD+ 2.20 ± 0.17 +NAD+ 1.01 ± 0.10 +Allopurinol 0.88 ± 0.19
94 ± 19
CNCh
−NAD+ 1.75 ± 0.49 +NAD+ 0.62 ± 0.19
nd
0.85 ± 0.17
Enzyme activities were assayed as described in Section 2. Results are expressed as mean ± S.D. (n = 15 for xanthine oxidase, n = 10 for catalase, n = 5 for SOD). nd: not determined. * p < 0.01 compared to non-chagasic sera (FBS, NCh, CNCh). # p < 0.01 compared to non-chagasic sera (FBS, NCh).
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Fig. 5. Effect of allopurinol, catalase and NEM on the growth of T. cruzi parasites exposed to different sera. 80 M allopurinol, 10 U/ml catalase, 200 M NEM and 10% (v/v) sera were added for 24 h. FBS: fetal bovine sera; ICh: indetermined chagasic sera; NCh: non-chagasic sera. Results are expressed as mean ± S.D. (n = 10 for each serum). p < 0.01 compared to the corresponding NCh and FBS. p < 0.01 compared to none ICh. None, allopurinol, catalase, allopurinol + catalase, NEM.
epimastigotes–, were used in order to support the role of XO and its product H2 O2 in the antiproliferative action of serum from chagasic patients. The addition of allopurinol (80 M) to the reaction mixture produced a 60% decrease in XOR activity (Table 1). Furthermore allopurinol increased parasite proliferation 65% when added to the growth medium containing chagasic sera. These effects of the XOR specific inhibitor indicate the participation of the enzyme in the antiproliferative action of chagasic sera. The addition of catalase (10 U/ml) to the medium containing chagasic sera resulted in a 100% increase in parasite proliferation. This result suggests the involvement of H2 O2 in the antiproliferative effect of chagasic sera. Allopurinol and catalase did not show additive effect when added jointly as growth rate did not differ from the values observed with catalase alone (Fig. 5) indicating that XO was not the only source of H2 O2 . When allopurinol or catalase were added to growth medium containing FBS or human sera from non-infected individuals, no significant difference in growth as compared with controls, was observed (Fig. 5). NEM concentration was selected in order to avoid any damage on the growth of the parasites grown in a medium supplemented with FBS. This selected NEM concentration did not affect parasite proliferation in the presence of human non-chagasic sera. The addition of NEM, however, resulted in a 50% elevation of the antiproliferative effect of chagasic sera (Fig. 5) which is in agreement with an increase in H2 O2 production. Serum catalase activity was measured in order to asses whether the XO mediated increase in H2 O2 production was affected by changes in the activity of this enzyme. Table 1 shows that catalase activity did not differ significantly between human sera from chagasic and non-chagasic individuals.
Determination of SOD activity in the parasites treated with the different sera showed that SOD activity was almost half as low in parasites grown in the presence of chagasic sera than in those incubated with FBS or normal human sera (Table 1) indicating a decrease in the defense mechanism against ROS. In conclusion these results suggest that the antiproliferative effect of chagasic sera could be due to oxidative stress. 4. Discussion It has been known since 1912 (Laveran and Mesnil, 1912) that normal human serum possesses innate protection against infection by the pathogen T. brucei brucei, but not against the agent of human
3.6. Sera from chagasic patients increase lipoperoxidation of T. cruzi epimastigotes membranes and decrease parasite SOD activity To determine whether an oxidative damage was involved in the trypanolytic action of the sera from chagasic patients, lipid peroxidation of polyunsaturated fatty acids of the parasite membranes was measured. Fig. 6 shows that there was a dose dependent increase in lipoperoxidation of the membranes of parasites treated with chagasic sera (Fig. 6). In contrast, an increase in FBS in the culture medium exerted a protective role against lipoperoxidation, while, at the highest assayed doses, normal human sera did not affect basal lipoperoxidation (Fig. 6).
Fig. 6. Effects of different sera on the lipoperoxidation of T. cruzi epimastigote membranes. Parasites were grown for 24 h in the presence of 10% (v/v) or 15% (v/v) of each serum. After this time parasites were harvested and homogenized to determine MDA levels. FBS: fetal bovine sera; ICh: indetermined chagasic sera; NCh: non-chagasic sera; CCh: chagasic patients with cardiopathy. Results are expressed as mean ± S.D. (n = 15 for each serum). p < 0.01 compared to FBS or NCh.
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African sleeping sickness, T. brucei rhodesiense. The observed protection is a result of non-immune killing factors present in human sera, known as trypanosome lytic factors (TLF). Two TLFs have been identified in human sera as circulating lipoprotein complexes, a subset of high density lipoprotein (HDL), which contain – in addition to apoA-1–, haptoglobin related protein, apolipoprotein L-1, and immunoglobulin M (Molina Portela et al., 2000; Raper et al., 2001; Vanhamme and Pays, 2004). Humans are susceptible to infection by T. cruzi. Nevertheless, human sera exhibit a trypanolytic action due to thermolabile factors one of which is related to the complement system and inhibits trypanosome epimastigote growth in vitro. The other is related to anti-alpha-galactosyl-antibodies which recognize the F2/3 antigenic fraction (Gazzinelli and Pereira, 1994; Almeida et al., 1994; Altcheh et al., 2003). The trypanolytic activity of normal human sera is abolished by heat inactivation at 56 ◦ C for 30 min. The data presented in this study show that sera from chagasic patients depleted of the complement system, displayed an antiproliferative action on T. cruzi epimastigotes, RA strain, when added to the growth medium, thereby indicating the presence of factors affecting the growth of the parasites other than those of the complement system. The antiproliferative effect was observed with sera of chagasic patients containing or depleted of specific antibodies, obtained from individuals without clinical manifestations or with cardiomyopathy. The antiproliferative effect was absent from the sera of uninfected healthy individuals and from uninfected patients with cardiovascular disease. Hence, restrained trypanosome growth may be due to the presence of growth inhibitory serum components other than specific antibodies or the complement system, induced by the infection and unrelated to the cardiomyopathy of the patient. The antiproliferative activity of sera from chagasic patients presented both concentration and time dependent behavior. A lytic effect was observed when the parasites were incubated with chagasic sera for 72 h at any of the assayed concentrations or at least for 24 h with 20% serum. Treatment induced parasite immobility, followed by complete growth arrest, loss of replication capacity upon transfer to fresh medium and eventual death. On the other hand, with serum doses below 20% or incubation times lower than 72 h, a trypanostatic action on the epimastigotes was observed; although the parasites stopped growing, they retained both their mobility and their capacity to proliferate when resuspended in a fresh medium. Neither trypanolytic nor trypanostatic effects were observed with FBS or with sera from uninfected individuals, suggesting that these effects were due to materials present in human serum produced post-infection. Sera were dialyzed as a first step towards the determination of the nature of the trypanolytic or trypanostatic serum component. The results obtained indicated that the antiproliferative material present in the chagasic serum was a non-dialyzable, high molecular weight substance. Taking into account that the antiproliferative effect was observed only in the sera of patients infected with T. cruzi and that XOR has been reported to participate in the systemic antimicrobial response of the innate immune system (Vorbach et al., 2003), we studied the activities of this enzyme in the different sera. Healthy individuals have low levels of circulating XOR (Martin et al., 2004). An increase in the enzyme activity in response to a range of diseases like endothelial dysfunction, hypertension, heart failure, and diabetes has been reported (Martin et al., 2004; Desco et al., 2002; Berry and Hare, 2004). In this study we report for the first time that the levels of XOR and XO increased considerably in all sera from both cardiac and indeterminate chagasic patients, presumably as a response to T. cruzi invasion. The infection elicits IFN-␥ production by natural killer cells and activates phagocytic cells, thus increasing hydrogen peroxide, nitric
oxide and TNF-␣ production (Cardoni, 1997; Samudio et al., 1998) all of which are essential for controlling acute parasitemia. XOR expression is stimulated by IFN-␥, IFN-␣ TNF-␣, IL-1 and IL-3. Some of these factors also initiate the conversion from XDH to XO, thus increasing ROS generation (Berry and Hare, 2004). It was reported that the Th1 pattern of immune response predominates during the entire course of the Chagas disease, including the chronic stage (Nabors and Tarleton, 1991; Cardoni et al., 1999; Antunez and Cardoni, 2000). This is consistent with our observations that both XOR and XO activities increased significantly in the serum of patients in the chronic phase of the disease. Although both laboratory and clinical investigations have reported an increase in the levels of endothelial bound XOR activity in a number of cardiovascular diseases (Berry and Hare, 2004), we observed that circulating XOR and XO activities in non-chagasic patients with dilated cardiopathy did not differ significantly from those of the control group (Table 1). The lack of increase in serum XOR activities in non-chagasic patients with dilated cardiopathy may be related to a different etiology of the disease, not involving oxidative stress. Lipid peroxidation is a major biomarker of ROS-generated oxidative damage. Increased lipid peroxidation of epimastigote membranes elicited by chagasic sera indicated the involvement of oxidative stress in the trypanolytic effect. The fact that sera of healthy individuals did not display an increase in XO activity or in ROS production, as evidenced by the maintenance of lipoperoxidation of parasite membranes at basal values, underlines the importance of the infection in triggering these processes. The antiproliferative effect of chagasic sera was decreased by both allopurinol – an inhibitor of XO–, and catalase – an enzyme that degrades hydrogen peroxide–. On the contrary, the addition of NEM, which by inhibiting hydrogen peroxide catabolism by the epimastigotes increases the levels of hydrogen peroxide, enhanced the antiproliferative activity. Taken together these results and our previous observation that the addition of hydrogen peroxide to the growth medium in the presence of NEM resulted in the death of the epimastigotes of T. cruzi (Hernandez et al., 2006), we conclude that the observed effects on parasite growth and viability are likely due to an increase in the concentration of hydrogen peroxide in the serum of the chagasic patients as a result of increase in XO activity. Moreover, this proposal is reinforced by the fact that SOD activity was much lower in parasites grown in the presence of chagasic sera since our previous studies and that of other authors demonstrated that the addition of hydrogen peroxide to the growth medium actually reduced the Fe/SOD activity in the epimastigote (Hernandez et al., 2006; Ismail et al., 1997). A consistent decline in Mn/SOD activity, the major oxygen radical scavenger in the mitochondrial matrix, during progression of infection and disease in chagasic myocardium has also been reported (Wen et al., 2004). Hydrogen peroxide can be generated by different types of oxidases. The fact that allopurinol, a specific inhibitor of XO, decreased the hydrogen peroxide dependent antiproliferative effect of chagasic sera, suggested that XO plays an important role in ROS generation in the sera of chagasic patients. Nevertheless we cannot discard the possible existence in the plasma of chagasic patients of other peroxide generating systems such as SOD and polyamineoxidases. In Cape Buffalo the natural resistance to T. brucei parasitemia has been attributed to hydrogen peroxide derived from serum XO. The increase in hydrogen peroxide correlates with a five to eightfold decline in blood catalase with no change in XO activity (Wang et al., 1999; Black et al., 2001). In contrast to the results obtained with Cape Buffalo, in the serum of chagasic patients at the chronic phase of the disease, we observed an increase in oxidative stress resulting from an increase in XO activity rather than from a decrease in catalase activity.
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In humans, sterilizing immunity does not appear to exist in T. cruzi infection. The XO dependent-ROS production triggered by the infection does not seem to have an antimicrobial role. Nevertheless, the continuous exposition to ROS thus generated, might contribute to the development of chagasic cardiomyopathy (Zacks et al., 2005). In conclusion, the data discussed so far provides evidence to support the idea that the antiproliferative activity observed in sera from chagasic patients may be due, among other factors, to a direct effect of hydrogen peroxide on the epimastigotes of T. cruzi. One of the greatest concerns in Chagas’ disease is the absence of reliable methods for the evaluation of chemotherapy efficacy in treated patients. The differences between sera of both infected and uninfected individuals shown in this study, may potentially be used to develop a reliable and safe test to follow both the evolution of the Chagas disease and the response to pharmacological treatment in patients. Acknowledgements Authors are very thankful to Dr. Elvira D. Isola and Dr. Estela Lammel from the Department of Microbiology, Parasitology and Inmunology (School of Medicine, University of Buenos Aires) for generously providing T. cruzi epimastigotes, to Dr. Alicia Fuchs for critical review and to Marianne Revah, Carolina Soleil and Melina Meneguin for language revision of the manuscript. This research was supported by a grant (PICTO 31428) from the Agencia Nacional de promoción Científica y Tecnológica and by the Universidad Abierta Interamericana. References Almeida, I.C., Ferguson, M.A.J., Schenkman, S., Travassos, L.R., 1994. Lytic anti␣-galactosil antibodies from patients with chronic Chagas disease recognize novel O-linked oligosaccharides on mucin-like glycosyl-phosphatidylinositolanchored glyproteins of Trypanosoma cruzi. Biochem. J. 304, 793–802. Altcheh, J., Corral, R., Biancardi, M., Freilij, H., 2003. Anticuerpos anti-F2/3 como ˜ con infección congénita por Trypanosoma cruzi. marcador de curación en ninos Medicina 63, 37–44. Antunez, M., Cardoni, R., 2000. IL-12 and INF ␥ production, and NK cell activity, in acute and chronic experimental Trypanosoma cruzi infections. Immunol. Lett. 71, 103–109. Berry, C.E., Hare, J.M., 2004. Xantine oxidoreductase and cardiovascular disease. Mechanism and physiological implications. J. Physiol. 555, 589–606. Black, S., Sicard, E., Murphy, N., Nolan, D., 2001. Innate and acquired control of trypanosome parasitaemia in Cape Buffalo. Int. J. Parasitol. 31, 562–565. Bradford, M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 7, 248–254. Cardoni, R., 1997. La respuesta inflamatoria en la infección aguda con Trypanosoma cruzi. Medicina 57, 227–234. Cardoni, R., Antunez, M., Abrami, A., 1999. Respuesta TH1 en la infección experimental con Trypanosoma cruzi. Medicina 59, 84–90. Cross, G.A.M., 1990. Cellular and genetic aspect of antigenic variation in Trypanosome. Annu. Rev. Immunol. 8, 83–110.
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