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The tumoricidal effect of Trypanosoma cruzi: its intracellular cycle and the immune response of the host
Medical Hypotheses (2000) 54(1), 1–6 © 2000 Harcourt Publishers Ltd Article No. mehy.1998.0808, available online at http://www.idealibrary.com on
The tumoricidal effect of Trypanosoma cruzi: its intracellular cycle and the immune response of the host H. R. A. Cabral Institute de Biología Celular, Facultad de Ciencias Médicas, Ciudad Universitaria, Córdoba, Argentina
Summary Many experimental evidences indicate that infection with Trypanosoma cruzi delays or inhibits the growth of malignant tumors in different strains of mice and in rats. These facts were verified by different workers. Although earlier workers proposed that this effect would be due to a toxin of T. cruzi, most of the accumulated evidences do not agree with such proposal. This present hypothesis agrees with the experimental data and proposes that the liberation of many endocellular antigens by destruction of some cancer cells, infected with T. cruzi, gives rise to an autoimmune response against antigens of analogous cancer cells, which limits or inhibits tumor growth. This point of view is supported by experimental studies on Chagas’ disease which showed the role of T. cruzi, to induce autoimmune reactions against target organs of the disease. On the basis of this hypothesis I postulate a new way to stimulate the immune system of the host against cancer. © 2000 Harcourt Publishers Ltd
INTRODUCTION In early experiments by Roskin and Exempliarskaja (1) it was found that a tumoricidal effect was produced in mice bearing a transplantable carcinoma when they were infected with Trypanosoma cruzi. This finding arose while they were investigating whether infectious agents, or toxins, could have effects on tumors. Roskin and coworkers (1–6) tried several infectious agents and toxins against several tumors but only when T. cruzi was used were significant and reproducible results obtained. Then, they postulated the existence of a substance from T. cruzi with toxic properties upon cancer cells and claimed to have obtained an endotoxin preparation (2,3), first, from plasma containing trypanosomes heated at 50°C to kill them and later on from an extract of lysed cells of T. cruzi (2–6). Although a report by Malisoff (7) agreed with them, another groups of workers tried to reproduce the experiments of Roskin and of Malisoff and concluded, in exhaustive studies, that endotoxin prepared according to Received 4 May 1998 Accepted 25 August 1998 Correspondence to: H. R. A. Cabral MD, PhD, Insituto de Biología Celular, Facultad de Ciencias Médicas, Ciudad Universitaria, 5000 Córdoba, Argentina. Fax: 054-351 4695101/4690047
Roskin and of Malisoff (2–6,7) was without effect against cancer (8–13). The lack of cancerolytic effect of killed T. cruzi preparations was observed in experiments either with cancerous animals or with cancer cell cultures (8–13). Therefore, I want to draw attention to the facts that the above-mentioned workers (1–6,8,9,13) and others (14–16) discovered with regard to the production of an antitumor effect when they infected with living T. cruzi their experimental cancer-bearing animals. HYPOTHESIS The tumoricidal action of T. cruzi could be due to autoimmune-type mechanisms against neoplastic cells, produced by the immune system of the tumor-bearing host itself, in response to certain effects of the active infection with this parasite. Living T. cruzi is able to invade some cancer cells and reproduce within them. Afterwards, their mature and motile forms breaks the cell membrane of the host cancer cell and goes to the extracellular space. Thus, the broken cancer cell leaves in the extracellular space its own cytoplasmic content and amastigote forms of T. cruzi, which dies in that place. Both biological materials remain exposed to the immune 1
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system of the host. In these conditions, a reaction against antigens of analogous cancer cells can be produced. In this process, the immature parasites could act as an adjuvant.
Trypanosoma cruzi infections: Characteristics in mammals Among the trypanosomes, T. cruzi is characterized by its reproductive cycle into mammalians, which occurs intracellularly in Leishmania-like forms (amastigote phase) (17–23). In a general manner, all eukariotic cells can be invaded by T. cruzi, but it prefers muscle cells – both neuroglial and neurones cells – and also reticuloendothelial cells (17–24). In vitro, the intracellular reproduction of T. cruzi has been demonstrated by employing different types of cultured cells, including malignant ones (19–28). When T. cruzi penetrates into the cell, it changes its shape, rounds up and, in this amastigote form, it reproduces over four or five days (20–26). At this time, some of them reach the trypanomastigote form, which becomes motile and destroys the cell membrane of the host cell (20–22,24–26). Thus, they reach the extracellular space. Maturation of intracellular parasites does not occur at the same time; many of them are still in their amastigote form, which dies in the extracellular space (20,21). It is remarkable that the invaded host cell tolerates well the invader parasites until they break its cell membrane, e.g. cultured cells extensively infected by T. cruzi are able to divide by mitosis and each daughter cell receive an equivalent charge of intracellular amastigote forms of T. cruzi (21). When living T. cruzi infects a tumor-bearing host, some malignant cells are invaded by the parasite, according to the results of several workers (1–5,8,9,13). This last fact indicates that T. cruzi have a moderate tropism for cancer cells. Does T. cruzi produce any toxin against mammalian cells? Dead T. cruzi have no effect on parenchymal cells of normal animals when they are injected, even in large amounts. Jörg (29) injected large doses of killed T. cruzi (from 0.01 g to 10 g/Kg body weight) into animals of various species. Under these conditions, neither acute nor chronic or delayed toxicity was observed (29). Besides, the strong survival of the normal and cancer cells in cultures extensively infected by living T. cruzi (17,21,22, 24–26,28) – with the exception of the cases of invaded and broken cells – would be convincing evidence against the existence of the toxic effect of this parasite. Cohen and coworkers (10), in a search for a carcinoclastic products, tested 3 trypanosomes (T. brasiliensis, T. lewisi and T. cruzi) which were ineffective in the test they chose, Medical Hypotheses (2000) 54(1), 1–6
which was the histologic condition of surviving tissue slice cultures from spontaneous carcinomas of Webster strain mice or transplantable Brown–Pearce carcinoma of rabbit (10). Belkin et al. (12) reported the absence of effects of lysed T. cruzi preparations on sarcoma 37 transplanted intramuscularly in CAF1 mice. Their histologic studies revealed no significant differences between dead T. cruzi-treated and tumor-bearing controls (12). Spain et al. (11) reported that no inhibition of tumor growth was noted in Bagg-albino strain mice with spontaneous mammary carcinoma, when treated with whole culture lysate of T. cruzi. Besides, microscopic lesions were neither found in histologic examinations of the tumors, nor in liver, kidney, intestine and heart of the animals which were treated with the lysates of T. cruzi when compared with the same types of tumor-bearing mice treated with sterile medium without T. cruzi (11).
Trypanosoma cruzi and the pathogeny of Chagas’ disease Chagas’ disease develops in man and in other susceptible mammals. Heart, both central and autonomic nervous system, and other organs with muscle and neural structures, such as the digestive tract, are affected. Vianna observed in 1911 (17) that many of the chagasic lesions did not contain T. cruzi and that the parasite are quite difficult to be found in the areas of active lesions. From this, some authors (30,31) postulated that both the circulating T. cruzi or its intracellular round form amastigote would produce a toxin, and this would be the cause of chagasic lesions. So far, most of the investigations concluded that neither the trypanomastigotes of T. cruzi nor the amastigote forms produced any toxin (8–11,20, 24–27,29,32,33). As mentioned, the host cells even invaded by the T. cruzi are able to reproduce by mitosis (19,20,24–27) and the parasites contained within them are transmitted to each daughter cells (20). These facts indicates that T. cruzi does not allect the behavior of the host cell until its cell membrane is broken by some mature young trypanosomes. The other reproductive forms of the parasite, still round and immature, die at the site of rupture of the host cell (intercellular space). As was pointed out above, T. cruzi has a special tropism by muscle, nervous and reticuloendothelial cells. In the course of Chagas’ chronic infection, infiltrates of mononuclear cells, mainly lymphocytes and monocyte-macrophages, are seen in affected organs (34). In this chronic stage of the Chagas’ disease, it is very difficult to find T. cruzi parasites in the target organs but they have cellular infiltrates of lymphocytes and other immunologic cells. It is worthwhile mentioning that the target organs of Chagas’ disease are composed predominantly of cells of the same types as those previously invaded by T. cruzi by its pref© 2000 Harcourt Publishers Ltd
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erential tropism (34). On the basis of the abovementioned facts, I postulate that many chagasic lesions would be produced by immunopathological reactivity, beginning when intracellular forms of T. cruzi complete their reproductive cycle and destroy some cell membranes in the host organism. The liberation of intracellular material could cause the immunocompetent system of the host to react against analogous cells disrupted by T. cruzi (34,35). Amastigote forms of T. cruzi, discharged together with the self components of the broken host cells, could act as an adjuvant (34). On the basis of this hypothesis, we experimentally transferred serum or leukocytes from mice infected with T. cruzi into uninfected mice of the same isogenic strain. Serum and leukocytes were previously treated to ensure that they contained no trypanosomes. Both receptor and infected mice showed several similar lesions, i.e. degeneration of neurones in the cerebral cortex, proliferation of neuroglia and oedema of the meninges, and lymphocytic infiltration in the heart, brain and skeletal muscles. On the other hand, lesions did not occur in control mice injected with serum or leukocytes of uninfected animals (35,36). Another event that can be related to the occurrence of autoimmunity in Chagas’ disease is the production of rheumatoid factors, in the acute and chronic stages of the human disease (37). Such factors were highly reactive with heterologous γ-globulin and were demonstrable with the Waaler–Rose reaction (37). Although it is possible that the rheumatoid factors produced during Chagas’ disease could be a natural protective response of the host to T. cruzi, it is also possible that these factors are autoantibodies, which could play a role in the autoimmunity in Chages’ disease, because of their reactivity with γ-globulin (37). Santos Buch and Teixeira (38) demonstrated in ‘in vitro’ experiments that lymphocytes from rabbits infected with T. cruzi were able to damage cardiac myocytes when they were co-cultivated. Teixeira and coworkers (39) showed that T-lymphocytes from chagasic patients had citotoxicity ‘in vitro’ both to human heart cells parasitized and non-parasitized by T. cruzi. Cossio et al. (40) found that lymphocytes from chagasic patients incubated ‘in vitro’ with murine or human heart preparations strongly adhered and interacted with the myocardial tissue. Laguens and colleagues (41) reported that they obtained transfer of cardiac lesions by means of spleen cells from mice chronically infected with T. cruzi when injected into uninfected mice of the same strain. On the other hand, in humans with chronic Chagas’ disease and with chagasic cardiopathy, we have found a T-lymphocyte subpopulation that intensely produced PAS+ substances and had CD4 receptors (35,42–44). These cells are found in a high number in circulating blood (more than 30% of total lymphocytes (35,42) and in some affected organs, such as heart (42–44). In this organ, © 2000 Harcourt Publishers Ltd
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the PAS+ T-lymphocytes were found around cardiomyocytes, which showed signs of damage, as well as around neural intracardiac structures (43,44). We performed a comparative study on the response of human lymphocytes from chagasic patients against human or murine heart or nervous tissues antigens and of the T. cruzi parasite, respectively (45,46). The lymphocytes of the chagasic patients underwent blastogenic transformation when treated with antigens of heart, brain and of the T. cruzi (in that order), with significant differences between the two former and the last one. The lymphocytes of healthy persons did not react significantly to any of these antigens. The obtained data enhanced the hypothesis which postulates (35,36,47) that, in Chagas’ disease, immunoreactivity is produced against antigens liberated by certain cells when their plasma membrane were broken due to the intracellular cycle of the T. cruzi (45). It is true that the data do not rule out completely the other hypothesis (48), which postulate that immunoreactivity could be produced because T. cruzi have antigens that would be shared by host tissues. However, according to the results of histologic studies of several workers (7–9,11,29) none of the organs usually affected by the chagasic process was damaged in the experimental animals injected with dead T. cruzi preparations. The above mentioned facts are also against the importance of cross-reactions in the Chagas’ disease.
Trypanosoma cruzi against tumors In their early experiments, Roskin and coworkers (1–5) observed that, in cancerous mice infected wit T. cruzi, the tumors decreased in size or even disappeared. This was confirmed by them with respect to Ehrlich carcinoma, Crocker sarcoma, cancer 63 and sarcoma 180 (1–5). Hauschka and coworkers (8,9) performed experiments in more than 1300 mice and several types of malignant tumors which were tested, both, in experiments with respect to infection with living T. cruzi, or with preparations derived from the parastie. Hauschka et al. (8,9) obtained negative results with their experiments about a cancerolytic substance prepared from dead T. cruzi following the techniques of Roskin et al. (2–5) and of Malisoff (7), respectively. Conversely, they found a cancerolytic effect limiting tumor growth, in experiments with living T. cruzi, injected inot other groups of mice of the same strains and with the same tumors (8,9). With regard to the A-mice strain with carcinoma 119, within 3 weeks of the beginning of the experiments, the average tumor volume was 450 mm3 in the test group as against 1300 mm3 in the control group (8). In addition, with implanted sarcoma 37, Hauschka and coworkers (8,9) found that in the test group infected with T. cruzi, the Medical Hypotheses (2000) 54(1), 1–6
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tumors reach a size of 226 mm3, as compared with 744 mm3 in the controls. In experiments in C3H mice with mammary adenocarcinoma, the tumor growth in the infected test group was also retarded, with an average size 252 mm3 against 554 mm3 in the control group, at 41 days (8,9). Hauschka et al. (8), by employing surviving mice of 3 months since infection with T. cruzi, assayed the effect on implanted tumors; in this test group, the tumors averaged 918 mm3 as against 1228 mm3 in the controls of comparable age without infection with T. cruzi. Jedeloo and coworkers (13) investigated the effect of dead T. cruzi, prepared according Roskin (4–6) and also Malisoff (7) on epidermal carcinoma of mice induced by skin painting with tar (13) and obtained negative results. If we re-examine the data reported by these workers (13) we observe that, in the group they infected with T. cruzi, the growth of tumors was significantly delayed, as compared with the other groups. Kagan et al. (15) also confirmed that living T. cruzi infection into female mice of high incidence of spontaneous mammary cancer, induced a significant retarded growth of the tumors. Knop and his colleagues (14) reported that an infection with living T. cruzi into mice with transplantable lymphoid leukemia exerted an inhibitory effect when the number of transplanted leukemic cells were less than 1.106. More recently, Oliveira and his coworkers (16) investigated the effect of 1,2 di-methylhydrazine -DMH-, a specific inducing colon cancer, on chronically T. cruziinfected rats. The animals were injected weekly for 12 weeks starting 100 days after the T. cruzi infection, and killed 6 months after starting DMH treatment. The incidence of colon adenocarcinoma in the groups infected with T. cruzi was 25.6% as against 65.6% in the group that received only DMH, with statistical significance between them (P < 0.001). Gaillard et al., in the early 1950s (49), reported experiments in human patients with cancer who were injected with living T. cruzi. The size of the tumors and pain decreased, but the survival was not prolonged (49). We think that, because of the inherent risk of Chagas’ disease, the inoculation of living T. cruzi into human patients would not be an advisable method of treatment. CONCLUSIONS A tumoricidal effect of infections with living T. cruzi has been observed by different groups of workers (1–5,8,9, 13–15), employing several strains of mice and rats, with transplantable and spontaneous tumors, who found that the growth of tumors was retarded or disappeared in some cases. The possibility of that the antitumoral effect of T. cruzi would be due to a toxic substance of the parasite was postulated and received some attention. Most of the Medical Hypotheses (2000) 54(1), 1–6
workers pointed out that preparations form dead T. cruzi, obtained through several experimental procedures, were without effect against transplantable or spontaneous tumors (8–13). At the same time, it was reported that such dead T. cruzi preparations had neither toxic effects nor histologic lesions of Chagas’ type in the organs of treated animals just as in in vitro experiments (8–13,29). The hypothesis presented here ascribes the tumoricidal effect induced by T. cruzi infection to the great area of the immune responses against cancer. This hypothesis takes into consideration the intracellular cycle of the T. cruzi in mammalians and points out their action in the liberation of cellular antigens of cancer cells into the extracellular space. Those biological materials remains expose to the immune system of the host and the also discharged immature parasite forms could act as an adjuvant. There is a general concept, as was previously widely established, about the importance of the immune system of the host which acts as natural defenses against malignant tumors and also there are experimental and clinical ways to stimulate the host immune system against tumors. On the basis of the above-mentioned concepts, and of the hypothesis here presented, I propose a way to obtain a preparation that could stimulate the host defenses against tumors, as follows: (1) Getting and breaking of fresh cancer cells from a given tumor; (2) Preparation of dead T. cruzi forms, obtained from parasite cultures; (3) Mixing of the fresh disrupted cancer cells with the killed T. cruzi; (4) Administration of such biological mixture (composed of fresh antigens of cancer cells and of T. cruzi), by injecting them into the same tumor-bearing host from which the samples of malignant tissue were obtained. I propose that such an experimental preparation could be assayed to induce or enhance the immune reactions against cancer. The suggested aspects could be useful both combined with the surgical treatment of a tumor, or with other forms of treatment of cancer.
ACKNOWLEDGEMENT The research described has partially supported by SE CYT, UNC, Córdoba, Argentina I thank Dr Alberto Candiotti for critical reading of the manuscript.
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4. Klyueva N. G., Roskin G. I. Cancerolytic substance of Schizotrypanum cruzi. Am Rev Soviet Med 1946; 4: 127–129. 5. Roskin G. I. Toxin therapy of experimental cancer. Cancer Res 1946; 6: 363–365. 6. Klyueva N. G. Paths of cancer biotherapy. Am Rev Soviet Med 1947; 4: 408–414. 7. Malisoff W. H. The action of the endotoxin of Trypanosoma cruzi (KR) on malignant mouse tumors. Science 1947; 106: 591–594. 8. Hauschka T. S., Saxe L. H. (jr), Blair M. Trypanosoma cruzi in treatment of muse tumors. J Nat Cancer Inst 1947; 7: 189–197. 9. Hauschka T. S., Goodwin M. B. Trypanosoma cruzi endotoxin (KR) in the treatment of malignant mouse tumors. Science 1948; 107: 600–602. 10. Cohen A. L., Borsook M., Dubnoff J. W. The effect of a Sporosarcina ureae preparation on tumor cells in vitro. Proc Soc Exper Biol Med 1947; 66: 440–444. 11. Spain D. M., Molomut N., Warshaw L. J. Preparations of lysates from cultures of T. cruzi and their effects on normal and tumor-bearing mice. Proc Soc Exper Biol Med 1948; 69: 134–136. 12. Belkin M., Tobie E. J., Kahler J., Shear M. J. Abscence of effect of lysed T. cruzi preparations on sarcoma 37. Cancer Res 1949; 9: 560. 13. Jedeloo G. C., Lignac G. O. E., Ligtenberg A. J., van Thiel P. H. J Natl Cancer Inst 1950; 10: 809–813. 14. Knop K., Hoecker G., Brncic D., Gasic G. Acción del Trypanosoma cruzi sobre tumores trasplantados del ratón. Bol Inform Parasitar Chil 1949; 4: 5–6. 15. Kagan I. G., Norman L., Hall E. C. The effect of infection with Trypanosoma cruzi on the development of spontaneous mammary cancer in mice. In: Anselmi A., ed. Medicina Tropical. Mexico: Edit Fournier 1968; 326–340. 16. Oliveira E. C., Miranda J. A. R., Leite M. S. B., Andrade A. L. S. S., Luquetti A. O., Moreira H. Lower incidence of drug-induced colon cancer in chronically Trypanosoma cruzi infected rats. Mem Inst Osw Cruz 1996; 91 Suppl 1: 317, 514a. 17. Vianna G. Contribuicao para o estudo da anatomia patolojica da ‘molestia de Carlos Chagas’. (Esquiso-tripanose humana ou tireoidite parazitaria). Mem Inst Osw Cruz 1911; 3: 276–294. 18. Dias E. Estudios sobre o Schizotrypanum cruzi. Mem Inst Osw Cruz 1934; 28: 1–110. 19. Romaña C., Meyer H. Estudo do ciclo evolutivo do Schizotrypanum cruzi em cultura de tecidos de embriao de galinha. Mem Inst Osw Cruz 1942; 37: 19–27. 20. Meyer H., Oliveira Musacchio M. X. Observacoes sobre divisoes mitoticas em células parasitadas. Ann Acad Brasil Cienc 1942; 14: 289–292. 21. Kofoid Ch A. The cycle of Trypanosoma cruzi in tissue culture of embrionic heart muscle. Univ Calif Publ Zoöl 1935; 41: 23–24. 22. Macedo V. Chagas’ disease (American Trypanosomiasis). In: Beeson P. B., McDermott W., Wyngaarden W. B., eds. Cecil Textbook of Medicine, 15th edn. Philadelphia: W. B. Saunders, 1979: 579–583. 23. Campos E. S. Studies upon a neurotropic strain of Trypanosoma cruzi. J Tech Meth Bull Internat Ass Med Mus 1929; 12: 146–147. 24. Villela E., Villela E. Elementos do sistema nervoso central parasitados pelo Trypanosoma cruzi. Mem Inst Osw Cruz 1932; 26: 77–81. 25. Meyer H., Oliveira Musacchio M. X. Sobre o desenvolvimento intracelular do Schizotrypanum cruzi em cultura de tecido como meio de verificacao da autonomia celular. Rev Brasil Biol 1944; 4: 81–86.
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26. Meyer H. Culturas de tecido nervoso infectadas por Schizotrypanum cruzi. Ann Acad Brasil Cienc 1942; 14: 253–256. 27. Rubio M. Mitosis en Células parasitadas por Trypanosoma cruzi Santiago Biológica, 1956: 51–61, Ch. 22. 28. Meyer H., Chagas Filho C. Cultivation of S. cruzi in tissue cultures of the spindle cell sarcoma ‘roffo’. Ann Acad Brasil Cienc 1950; 22: 175–182. 29. Jörg M. E. Imposibilidad de demostrar toxinas en Trypanosoma cruzi de cultivos. Bol Chileno Paras 1964; 19: 84–87. 30. Mayer M., Rocha Lima H. Zum verhalten von Schizotrypanum cruzi in Warmblütern und Arthropoden. Arch Schiffs- u TropenHyg 1914; 18: 101–136. 31. Köberle F. Ueber das Neurotoxin des Trypanosoma cruzi. Zentrabl Allg Path u Path Anat 1956; 95: 468–475. 32. Eichbaum F. W. Pesquisas sobre a presenca de substancias tóxicas em culturas de Trypanosoma cruzi. Ann Cong Internac Doenca Chagas (Rio de Janeiro) 1959; 2: 479–489. 33. Oliveira Musacchio M. X., Meyer H. Acao do Schizotrypanum cruzi degenerado ou em suspensao de tripanosomas mortos sobre células nervosas em culturas de tecido de embriao de galinha. Hospital 1959; 55: 899–903. 34. Cabral H. R. A. Los mecanismos patogenéticos del daño tisular en la enfermedad de Chagas. Rer Fac Cienc Méd Córdoba 1969; 27: 287–309. 35. Cabral H. R. A. PAS-positive substance in lymphocytes of patients with Chagas’ disease. Lancet 1971; 1: 1356–1357. 36. Cabral H. R. A., Segura-Seco E. L., Paolasso E. W., Castoldi F., Veloso M., Cichero J. Enfermedad de Chagas aguda y autoinmunidad. (Hipótesis sobre la patogenia de la injuria tisular e intento de verificación experimental). Rev Fac Cienc Méd Córdoba 1967; 25: 419–431. 37. Cabral H. R. A. Rheumatoid factors and Chagas’ disease. Science 1983; 219: 1238. 38. Santos-Buch Ch A., Teixeira A. R. L. The immunology of experimental Chagas’ disease. III. Rejection of allogeneic heart cells in vitor. J Exp Med 1974; 140: 38–53. 39. Teixeira A. R. L., Teixeira G., Macedo V., Prata A. Trypanosoma cruzi-sensitized T-lymphocyte mediated 51Cr release from human heart cells in Chagas’ disease. Am J Trop Med Hyg 1978; 27: 1097–1107. 40. Cossio P., Damilano G., Dela Vega M. T., Laguens R. F., Cabeza Meckert P., Diez C., Arana R. M. In vitro interaction between lymphocytes of chagasic individuals and heart tissue. Medicina 1976; 36: 287–293. 41. Laguens P., Cabeza Meckert P., Gelpi R., Chambó J. G. Transferencia de lesiones cardíacas por medio de células esplénicas de ratones con enfermedad de Chagas crónica. Medicina 1980; 40: 808–809. 42. Cabral H. R. A., Braxs J. Producción de una glicoproteína por linfocitos T en la cardiopatía chagásica. Medicina 1982; 42: 415–421. 43. Cabral H. R. A., Novak I. Immunohistochemical staining for characterization of glycoprotein producing T-lymphocytes in chagasic heart disease. Mem Inst Osw Cruz 1995; 90: Suppl 1: 158. 44. Cabral H. R. A., Novak I. T. C., Robert G. B. Comprobación de vénulas de endotelio alto en corazones de pacientes chagásicos con cardiomiopatía crónica grave. Rev Argent Cardiol 1993; 61: 463–465. 45. Cabral H. R. A., Braxs J., Mendez M. T. Transformación blástica de linfocitos de chagásicos frente a antígenos de corazón y de cerebro. Rev Méd Córdoba 1990; 78: 16–18. 46. Sanz L. M., Cabral H. R. A. Blastic transformation of lymphocytes from chagasic patients cultured in vitro and challenged with human heart antigens. Commun Biol 1987; 6: 165.
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The tumoricidal effect of Trypanosoma cruzi: its intracellular cycle and the immune response of the host H. R. A. Cabral Institute de Biología Celular, Facultad de Ciencias Médicas, Ciudad Universitaria, Córdoba, Argentina
Summary Many experimental evidences indicate that infection with Trypanosoma cruzi delays or inhibits the growth of malignant tumors in different strains of mice and in rats. These facts were verified by different workers. Although earlier workers proposed that this effect would be due to a toxin of T. cruzi, most of the accumulated evidences do not agree with such proposal. This present hypothesis agrees with the experimental data and proposes that the liberation of many endocellular antigens by destruction of some cancer cells, infected with T. cruzi, gives rise to an autoimmune response against antigens of analogous cancer cells, which limits or inhibits tumor growth. This point of view is supported by experimental studies on Chagas’ disease which showed the role of T. cruzi, to induce autoimmune reactions against target organs of the disease. On the basis of this hypothesis I postulate a new way to stimulate the immune system of the host against cancer. © 2000 Harcourt Publishers Ltd
INTRODUCTION In early experiments by Roskin and Exempliarskaja (1) it was found that a tumoricidal effect was produced in mice bearing a transplantable carcinoma when they were infected with Trypanosoma cruzi. This finding arose while they were investigating whether infectious agents, or toxins, could have effects on tumors. Roskin and coworkers (1–6) tried several infectious agents and toxins against several tumors but only when T. cruzi was used were significant and reproducible results obtained. Then, they postulated the existence of a substance from T. cruzi with toxic properties upon cancer cells and claimed to have obtained an endotoxin preparation (2,3), first, from plasma containing trypanosomes heated at 50°C to kill them and later on from an extract of lysed cells of T. cruzi (2–6). Although a report by Malisoff (7) agreed with them, another groups of workers tried to reproduce the experiments of Roskin and of Malisoff and concluded, in exhaustive studies, that endotoxin prepared according to Received 4 May 1998 Accepted 25 August 1998 Correspondence to: H. R. A. Cabral MD, PhD, Insituto de Biología Celular, Facultad de Ciencias Médicas, Ciudad Universitaria, 5000 Córdoba, Argentina. Fax: 054-351 4695101/4690047
Roskin and of Malisoff (2–6,7) was without effect against cancer (8–13). The lack of cancerolytic effect of killed T. cruzi preparations was observed in experiments either with cancerous animals or with cancer cell cultures (8–13). Therefore, I want to draw attention to the facts that the above-mentioned workers (1–6,8,9,13) and others (14–16) discovered with regard to the production of an antitumor effect when they infected with living T. cruzi their experimental cancer-bearing animals. HYPOTHESIS The tumoricidal action of T. cruzi could be due to autoimmune-type mechanisms against neoplastic cells, produced by the immune system of the tumor-bearing host itself, in response to certain effects of the active infection with this parasite. Living T. cruzi is able to invade some cancer cells and reproduce within them. Afterwards, their mature and motile forms breaks the cell membrane of the host cancer cell and goes to the extracellular space. Thus, the broken cancer cell leaves in the extracellular space its own cytoplasmic content and amastigote forms of T. cruzi, which dies in that place. Both biological materials remain exposed to the immune 1
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system of the host. In these conditions, a reaction against antigens of analogous cancer cells can be produced. In this process, the immature parasites could act as an adjuvant.
Trypanosoma cruzi infections: Characteristics in mammals Among the trypanosomes, T. cruzi is characterized by its reproductive cycle into mammalians, which occurs intracellularly in Leishmania-like forms (amastigote phase) (17–23). In a general manner, all eukariotic cells can be invaded by T. cruzi, but it prefers muscle cells – both neuroglial and neurones cells – and also reticuloendothelial cells (17–24). In vitro, the intracellular reproduction of T. cruzi has been demonstrated by employing different types of cultured cells, including malignant ones (19–28). When T. cruzi penetrates into the cell, it changes its shape, rounds up and, in this amastigote form, it reproduces over four or five days (20–26). At this time, some of them reach the trypanomastigote form, which becomes motile and destroys the cell membrane of the host cell (20–22,24–26). Thus, they reach the extracellular space. Maturation of intracellular parasites does not occur at the same time; many of them are still in their amastigote form, which dies in the extracellular space (20,21). It is remarkable that the invaded host cell tolerates well the invader parasites until they break its cell membrane, e.g. cultured cells extensively infected by T. cruzi are able to divide by mitosis and each daughter cell receive an equivalent charge of intracellular amastigote forms of T. cruzi (21). When living T. cruzi infects a tumor-bearing host, some malignant cells are invaded by the parasite, according to the results of several workers (1–5,8,9,13). This last fact indicates that T. cruzi have a moderate tropism for cancer cells. Does T. cruzi produce any toxin against mammalian cells? Dead T. cruzi have no effect on parenchymal cells of normal animals when they are injected, even in large amounts. Jörg (29) injected large doses of killed T. cruzi (from 0.01 g to 10 g/Kg body weight) into animals of various species. Under these conditions, neither acute nor chronic or delayed toxicity was observed (29). Besides, the strong survival of the normal and cancer cells in cultures extensively infected by living T. cruzi (17,21,22, 24–26,28) – with the exception of the cases of invaded and broken cells – would be convincing evidence against the existence of the toxic effect of this parasite. Cohen and coworkers (10), in a search for a carcinoclastic products, tested 3 trypanosomes (T. brasiliensis, T. lewisi and T. cruzi) which were ineffective in the test they chose, Medical Hypotheses (2000) 54(1), 1–6
which was the histologic condition of surviving tissue slice cultures from spontaneous carcinomas of Webster strain mice or transplantable Brown–Pearce carcinoma of rabbit (10). Belkin et al. (12) reported the absence of effects of lysed T. cruzi preparations on sarcoma 37 transplanted intramuscularly in CAF1 mice. Their histologic studies revealed no significant differences between dead T. cruzi-treated and tumor-bearing controls (12). Spain et al. (11) reported that no inhibition of tumor growth was noted in Bagg-albino strain mice with spontaneous mammary carcinoma, when treated with whole culture lysate of T. cruzi. Besides, microscopic lesions were neither found in histologic examinations of the tumors, nor in liver, kidney, intestine and heart of the animals which were treated with the lysates of T. cruzi when compared with the same types of tumor-bearing mice treated with sterile medium without T. cruzi (11).
Trypanosoma cruzi and the pathogeny of Chagas’ disease Chagas’ disease develops in man and in other susceptible mammals. Heart, both central and autonomic nervous system, and other organs with muscle and neural structures, such as the digestive tract, are affected. Vianna observed in 1911 (17) that many of the chagasic lesions did not contain T. cruzi and that the parasite are quite difficult to be found in the areas of active lesions. From this, some authors (30,31) postulated that both the circulating T. cruzi or its intracellular round form amastigote would produce a toxin, and this would be the cause of chagasic lesions. So far, most of the investigations concluded that neither the trypanomastigotes of T. cruzi nor the amastigote forms produced any toxin (8–11,20, 24–27,29,32,33). As mentioned, the host cells even invaded by the T. cruzi are able to reproduce by mitosis (19,20,24–27) and the parasites contained within them are transmitted to each daughter cells (20). These facts indicates that T. cruzi does not allect the behavior of the host cell until its cell membrane is broken by some mature young trypanosomes. The other reproductive forms of the parasite, still round and immature, die at the site of rupture of the host cell (intercellular space). As was pointed out above, T. cruzi has a special tropism by muscle, nervous and reticuloendothelial cells. In the course of Chagas’ chronic infection, infiltrates of mononuclear cells, mainly lymphocytes and monocyte-macrophages, are seen in affected organs (34). In this chronic stage of the Chagas’ disease, it is very difficult to find T. cruzi parasites in the target organs but they have cellular infiltrates of lymphocytes and other immunologic cells. It is worthwhile mentioning that the target organs of Chagas’ disease are composed predominantly of cells of the same types as those previously invaded by T. cruzi by its pref© 2000 Harcourt Publishers Ltd
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erential tropism (34). On the basis of the abovementioned facts, I postulate that many chagasic lesions would be produced by immunopathological reactivity, beginning when intracellular forms of T. cruzi complete their reproductive cycle and destroy some cell membranes in the host organism. The liberation of intracellular material could cause the immunocompetent system of the host to react against analogous cells disrupted by T. cruzi (34,35). Amastigote forms of T. cruzi, discharged together with the self components of the broken host cells, could act as an adjuvant (34). On the basis of this hypothesis, we experimentally transferred serum or leukocytes from mice infected with T. cruzi into uninfected mice of the same isogenic strain. Serum and leukocytes were previously treated to ensure that they contained no trypanosomes. Both receptor and infected mice showed several similar lesions, i.e. degeneration of neurones in the cerebral cortex, proliferation of neuroglia and oedema of the meninges, and lymphocytic infiltration in the heart, brain and skeletal muscles. On the other hand, lesions did not occur in control mice injected with serum or leukocytes of uninfected animals (35,36). Another event that can be related to the occurrence of autoimmunity in Chagas’ disease is the production of rheumatoid factors, in the acute and chronic stages of the human disease (37). Such factors were highly reactive with heterologous γ-globulin and were demonstrable with the Waaler–Rose reaction (37). Although it is possible that the rheumatoid factors produced during Chagas’ disease could be a natural protective response of the host to T. cruzi, it is also possible that these factors are autoantibodies, which could play a role in the autoimmunity in Chages’ disease, because of their reactivity with γ-globulin (37). Santos Buch and Teixeira (38) demonstrated in ‘in vitro’ experiments that lymphocytes from rabbits infected with T. cruzi were able to damage cardiac myocytes when they were co-cultivated. Teixeira and coworkers (39) showed that T-lymphocytes from chagasic patients had citotoxicity ‘in vitro’ both to human heart cells parasitized and non-parasitized by T. cruzi. Cossio et al. (40) found that lymphocytes from chagasic patients incubated ‘in vitro’ with murine or human heart preparations strongly adhered and interacted with the myocardial tissue. Laguens and colleagues (41) reported that they obtained transfer of cardiac lesions by means of spleen cells from mice chronically infected with T. cruzi when injected into uninfected mice of the same strain. On the other hand, in humans with chronic Chagas’ disease and with chagasic cardiopathy, we have found a T-lymphocyte subpopulation that intensely produced PAS+ substances and had CD4 receptors (35,42–44). These cells are found in a high number in circulating blood (more than 30% of total lymphocytes (35,42) and in some affected organs, such as heart (42–44). In this organ, © 2000 Harcourt Publishers Ltd
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the PAS+ T-lymphocytes were found around cardiomyocytes, which showed signs of damage, as well as around neural intracardiac structures (43,44). We performed a comparative study on the response of human lymphocytes from chagasic patients against human or murine heart or nervous tissues antigens and of the T. cruzi parasite, respectively (45,46). The lymphocytes of the chagasic patients underwent blastogenic transformation when treated with antigens of heart, brain and of the T. cruzi (in that order), with significant differences between the two former and the last one. The lymphocytes of healthy persons did not react significantly to any of these antigens. The obtained data enhanced the hypothesis which postulates (35,36,47) that, in Chagas’ disease, immunoreactivity is produced against antigens liberated by certain cells when their plasma membrane were broken due to the intracellular cycle of the T. cruzi (45). It is true that the data do not rule out completely the other hypothesis (48), which postulate that immunoreactivity could be produced because T. cruzi have antigens that would be shared by host tissues. However, according to the results of histologic studies of several workers (7–9,11,29) none of the organs usually affected by the chagasic process was damaged in the experimental animals injected with dead T. cruzi preparations. The above mentioned facts are also against the importance of cross-reactions in the Chagas’ disease.
Trypanosoma cruzi against tumors In their early experiments, Roskin and coworkers (1–5) observed that, in cancerous mice infected wit T. cruzi, the tumors decreased in size or even disappeared. This was confirmed by them with respect to Ehrlich carcinoma, Crocker sarcoma, cancer 63 and sarcoma 180 (1–5). Hauschka and coworkers (8,9) performed experiments in more than 1300 mice and several types of malignant tumors which were tested, both, in experiments with respect to infection with living T. cruzi, or with preparations derived from the parastie. Hauschka et al. (8,9) obtained negative results with their experiments about a cancerolytic substance prepared from dead T. cruzi following the techniques of Roskin et al. (2–5) and of Malisoff (7), respectively. Conversely, they found a cancerolytic effect limiting tumor growth, in experiments with living T. cruzi, injected inot other groups of mice of the same strains and with the same tumors (8,9). With regard to the A-mice strain with carcinoma 119, within 3 weeks of the beginning of the experiments, the average tumor volume was 450 mm3 in the test group as against 1300 mm3 in the control group (8). In addition, with implanted sarcoma 37, Hauschka and coworkers (8,9) found that in the test group infected with T. cruzi, the Medical Hypotheses (2000) 54(1), 1–6
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tumors reach a size of 226 mm3, as compared with 744 mm3 in the controls. In experiments in C3H mice with mammary adenocarcinoma, the tumor growth in the infected test group was also retarded, with an average size 252 mm3 against 554 mm3 in the control group, at 41 days (8,9). Hauschka et al. (8), by employing surviving mice of 3 months since infection with T. cruzi, assayed the effect on implanted tumors; in this test group, the tumors averaged 918 mm3 as against 1228 mm3 in the controls of comparable age without infection with T. cruzi. Jedeloo and coworkers (13) investigated the effect of dead T. cruzi, prepared according Roskin (4–6) and also Malisoff (7) on epidermal carcinoma of mice induced by skin painting with tar (13) and obtained negative results. If we re-examine the data reported by these workers (13) we observe that, in the group they infected with T. cruzi, the growth of tumors was significantly delayed, as compared with the other groups. Kagan et al. (15) also confirmed that living T. cruzi infection into female mice of high incidence of spontaneous mammary cancer, induced a significant retarded growth of the tumors. Knop and his colleagues (14) reported that an infection with living T. cruzi into mice with transplantable lymphoid leukemia exerted an inhibitory effect when the number of transplanted leukemic cells were less than 1.106. More recently, Oliveira and his coworkers (16) investigated the effect of 1,2 di-methylhydrazine -DMH-, a specific inducing colon cancer, on chronically T. cruziinfected rats. The animals were injected weekly for 12 weeks starting 100 days after the T. cruzi infection, and killed 6 months after starting DMH treatment. The incidence of colon adenocarcinoma in the groups infected with T. cruzi was 25.6% as against 65.6% in the group that received only DMH, with statistical significance between them (P < 0.001). Gaillard et al., in the early 1950s (49), reported experiments in human patients with cancer who were injected with living T. cruzi. The size of the tumors and pain decreased, but the survival was not prolonged (49). We think that, because of the inherent risk of Chagas’ disease, the inoculation of living T. cruzi into human patients would not be an advisable method of treatment. CONCLUSIONS A tumoricidal effect of infections with living T. cruzi has been observed by different groups of workers (1–5,8,9, 13–15), employing several strains of mice and rats, with transplantable and spontaneous tumors, who found that the growth of tumors was retarded or disappeared in some cases. The possibility of that the antitumoral effect of T. cruzi would be due to a toxic substance of the parasite was postulated and received some attention. Most of the Medical Hypotheses (2000) 54(1), 1–6
workers pointed out that preparations form dead T. cruzi, obtained through several experimental procedures, were without effect against transplantable or spontaneous tumors (8–13). At the same time, it was reported that such dead T. cruzi preparations had neither toxic effects nor histologic lesions of Chagas’ type in the organs of treated animals just as in in vitro experiments (8–13,29). The hypothesis presented here ascribes the tumoricidal effect induced by T. cruzi infection to the great area of the immune responses against cancer. This hypothesis takes into consideration the intracellular cycle of the T. cruzi in mammalians and points out their action in the liberation of cellular antigens of cancer cells into the extracellular space. Those biological materials remains expose to the immune system of the host and the also discharged immature parasite forms could act as an adjuvant. There is a general concept, as was previously widely established, about the importance of the immune system of the host which acts as natural defenses against malignant tumors and also there are experimental and clinical ways to stimulate the host immune system against tumors. On the basis of the above-mentioned concepts, and of the hypothesis here presented, I propose a way to obtain a preparation that could stimulate the host defenses against tumors, as follows: (1) Getting and breaking of fresh cancer cells from a given tumor; (2) Preparation of dead T. cruzi forms, obtained from parasite cultures; (3) Mixing of the fresh disrupted cancer cells with the killed T. cruzi; (4) Administration of such biological mixture (composed of fresh antigens of cancer cells and of T. cruzi), by injecting them into the same tumor-bearing host from which the samples of malignant tissue were obtained. I propose that such an experimental preparation could be assayed to induce or enhance the immune reactions against cancer. The suggested aspects could be useful both combined with the surgical treatment of a tumor, or with other forms of treatment of cancer.
ACKNOWLEDGEMENT The research described has partially supported by SE CYT, UNC, Córdoba, Argentina I thank Dr Alberto Candiotti for critical reading of the manuscript.
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