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The effect of BCG on the course of experimental Chagas' disease in mice
THE EFFECT OF BCG ON THE COURSE OF EXPERIMENTAL CHAGAS’ DISEASE IN MICE R. E. KUHN, R. T. VAUGHN and G. A. HERBST Department
of Biology, Wake Forest University, Winston-Salem, (Received
17 December
North Carolina 27109, U.S.A.
1974)
Abstract-KUHN R. E., VAUGHNR. T. and HERBST G. A. 1975. The effect of BCG on the course of experimental Chagas’ disease in mice. international Jo14rnaE for ParasitoZogy 5: 557-560. Experiments were done to determine the effect of BCG treatment on longevity, development of parasitemia, and in vivo distribution of 51Cr-labelled trypanosomes in C3H(He) female mice infected with a Brazil strain of Trypanosoma cruzi. BCG sensitization of mice was accomplished by a single IV injection of 3.0 mg (wet weight) of BCG. Twenty-one days after BCG injection mice were infected with 5 x IO" blood-form trypomastigotes. Parasitemia dete~ina~i~ns were made on alternate days during the experiment while in vivo distribution of exogenously supplied 51Cr~pimastigotes was made in groups of BCG or PBS stimulated mice on day 15 of the T. cruzi infection. It was found that BCG sensitization had no effect on longevity or parasitemia development in T. cvuzi infected C3H(He) female mice, There were, however, some differences in the in vivo distribution of parasites between BCG treated and control mice. BCG stimulated mice accumulated greater numbers of radiolabelled trypanosomes in the kidneys and small intestines while PBS treated mice were found to have greater numbers of labelled parasites in the liver. Although no significant differences were observed in longevity of BCG or PBS treated mice, it was noted that BCG treated animals which were bled for parasitemia dete~inations lived significantly longer than those which were merely observed for longevity. INDEX KEY WORDS: Trypanosoma
cruzi; BCG; immunity;
THE ROLE
of the macrophage in acute Trypanosoma in mammals is not understood. Taliaferro & Pizzi (1955) found in in viva studies that macrophages can destroy these trypanosomes but that, in some cases, the parasite proliferates in macrophages and may be responsible for depletion of reticuloendothelial elements during the course of acute experimental Chagas’ disease. Behbehani (1973) later reported that, although the parasite was often killed by macrophages in vitro, in many cases amastigotes proliferated intracellularly and were capable of transfo~ing into t~pom~tigotes which could escape the macrophage to reinvade other cells. Dvorak & Schmunis (1972), on the other hand, convincingly showed that phagocytosed epimastigotes and trypomastigote forms of the parasite were killed by macrophages irz v&o and that the parasite was incapable of avoiding phagocytic destruction. In viva experiments have shown that manipulation of the reticul~ndothelial system (RES) can affect the course of experimental Chagas’ disease. Kierszenbaum, Knecht, Budzko & Pizzimenti (1974) have shown that stimulation of the RES by diethylstilbestrol results in greater longevity, reduced mortality, and decreased parsitemia in T. cruzi infected mice. cruzi
infections
557
Wr-Iabelling;
Chagas’ disease; mice.
These workers also confirmed earlier studies showing that depression of the RES results in increased severity of the disease (Goble & Boyd, 1962). The destructive capacity of macrophages may play a primary role in immune protection against many eucaryotic protozoan parasitic diseases. Enhancing macrophage activity, indeed, may be impo~ant in i~unothera~utic means of treating some of these diseases. Beneficial effects of non-specific stimulation of the immune system as reported by Kierszenbaum et al. (1974) tends to support this claim. An immune stimulant which has not been examined in a protozoan parasite system is the B&flus Camette-Guerin (BCG} which is an attenuated strain of tubercle bacilli. BCG has been extensively investigated as an immunotherapeutic agent in human and animal tumor systems and has revealed a non-specific BCG-induced regression of several tumor types (Lemond, 1973; Faraci & Schour, 1974). It has recently been demonstrated that, in addition to non-specific stimulation, BCGtreated mice may also develop an enhanced specific cytotoxicity to some experimental tumors (Faraci &. Schour, 1974). On the basis of the success of BCG stimulation 01 cell-mediated responses in some tumor systems and the effectiveness of diethystilbestrol stimulation in
558
R. E.
KUHN, R. T. VAUGHN
experimental Chagas’ disease, it seems appropriate to examine the effect of BCG on the course of T. cruzi infections in mice. MATERIALS Animals.
AND
METHODS
All mice in this study were C3H(He) females
weighing 18-20 g and were purchased from Flow Research Laboratories, Inc. (Dublin, Va.). Animals were maintained in our laboratory for two weeks prior to use in experiments and were housed 6 to a cage. Purina rat chow and water were supplied ad libitum. Infections. Mice were infected bv intraperitoneal (IP) injection of 5 x lo4 trypomastigotes (a Biazil strain) in 0.1 ml of ohosnhate buffered saline (PBS). Trvnomastigates wer; taken from stock mice‘ which &d been infected with 5 x IO* trypomastigotes 14 days earlier. Donor mice were bled from the orbital plexus into cold Alsevers’ solution and the blood subsequently centrifuged and washed three times in cold PBS. The concentration of parasites was determined by hemacytometer counting and adjusted to 5 x lo4 trypomastigotes per 0.1 ml of PBS. C3H(He) female mice (20 g) receiving this inoculum usually die on day 17 or 18. Parasitemia and longevity determinations. Separate groups of mice were used to determine the effect of BCG on parasitemia and longevity. Ten experimental and 10 control mice were used for parasitemia studies and two other groups of 10 mice were used for longevity determinations. Parasitemia in infected animals was determined by placing a 4~1 sample of tail vein blood uniformly under an 18 mm circular glass coverslip, counting the parasites in 100 fields, and converting this figure to the number of parasites per ml of blood. Parasitemia determinations were made on alternate days beginning with day 8 of the infection period. Cages were checked each morning and each afternoon for dead mice. Animals found dead in the afternoon were considered as having half-day increased longevity to animals found dead in the morning. Bacillus Calmette-Guerin (BCG). Cultures of BCG were obtained from the laboratory of Dr. Quentin Myrvik, Department of Microbiology, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, North Carolina. BCG was grown in Dubose medium (Difco Laboratories, Detroit: Michigan) at 37°C for 21 days. The bacilli were then centrifuged at 3000 rev./min for 10 min, washed with 1.0 ml PBS and weighed in tared test tubes. The cultures were resuspended-with a Broeck-tvoe tissue grinder in sufficient PBS to contain 3 mg Bdb (wet w$O.2 ml PBS. This inoculum (3 mg) was injected IV into the experimental animals. Twentyone days after BCG injection (or PBS for controls), mice were infected with 5 x lo4 trypomastigotes. In vivo distribution. The in vivo distribution of SICrlabelled culture-forms of T. cruzi was determined in BCG and PBS treated mice on day 15 of the trypanosome infection by the method of Kuhn, Vaughn & Iannuzzi (1974). Four hours after experimental mice were injected IV with 10 x lo8 labelled trypanosomes, they were bled to death from the orbital plexus and the following organs removed: liver, spleen, kidneys, heart, lungs, skeletal muscle (right upper hindleg musculature), brain, axillary and bra&al lymph nodes, and skin. The blood and
and G. A.
HERBST
I.J.P. VOL.
5, 1975
organs were then counted for radioactivity in a Nuclear Chicago well scintillation counter and the data calculated using ?he following formula: c.p.m. per specified organ x 100. total recovered c.p.m. Statistics. Data were evaluated using the Student t-test and are presented with standard deviations where applicable. RESULTS
The development of parasitemia in PBS and BCG treated mice is shown in Fig. 1. Parasitemias in the
two groups parallelled each other until day 20 after which time the BCG treated animals developed and
. / .,.;’
24 22 20
0
0 u ‘il . E
16 I6
-
I
BCG experimentols PBS controls
14
E 12 Cl S 10 g 5 I-
6 6 4
Day
of
InfectIon
FIG. I. Parasitemia values for BCG and PBS treated mice during the course of experimental Chagas’ disease.
sustained greater parasitemias. The higher parasitemia values recorded for the BCG treated mice, however, are not significantly different (P < 0.05) from the control values at any time during the course of infection. Mean longevity for the two groups of mice used for parasitemia determinations was 26.8 & 2.7 and 25.7 * 7.59 for the BCG and PBS treated groups, respectively. Table 1 contains information regarding the longevity of BCG treated and control mice. As can TABLE I-LONGEVITY
IN BCG AND PBS TREATEDC3H(He) FEMALE MICE*-i_ Mean survival
Treatment
BCG longevity group PBS longevity group BCG parasitemia group PBS parasitemia group
(davs) i,
23.2 23.4 26.8 25.7
f f f *
I.64 5.26 2.7 7.59
Ranee
20-25 15-28 20-30 18-39
* N = IO for all groups. t Longevity groups were not bled for parasitemia determinations.
be seen, the mean survival time of the BCG group, (23.2 + , _ 1.64), was not different from control
I.J.P. VOL. 5.
1975
Immunity to T~ypano~oma cruzi
animals (23.4 & 5.26). Survival times ranged from 20 to 25 days for the BCG treated mice while the control mice began dying on day 15 with 100% mortality not occurring until day 28. There were, however, some statistically significant differences in the in vivo distribution of 51-Cr-labelled trypanosomes between the two groups. Table 2 presents the data as % accumulation of label in TABLE 2--h vivo DISTRIBUTION OF TRYPANOSOMES IN BCG AND
PBS
TREATEDC3H(He)
Organ or tissue Blood Liver 0 Spleen Kidneys 0 Small intestine* Heart Lungs Skeletal muscle Brain Lymph nodes Skin
FEMALEMICE*‘~$
BCG treated 5.23 51.88 7.80 7.17 2.65 0.51 15.25 1.51 0.65 0.35 7.00
* 1.77 jr 3.27 * 1.62 i 1.39 f 0.82 i 0.44 ri_ 2.46 t 0.91 i 0.60 + 0.40 f 1.69
PBS controls 4.22 58.02 7.53 4.74 1.76 0.55 14.22 0.95 0.57 0.38 6.89
i 1.35 + 4.40 + 1.56 & 1.18 + 0.96 f 0.59 * 3.12 f 0.80 1 0.80 i 0.42 !L 2.52
*All values expressed as “/” recovered c.D.m. tFor BCG group n = 11 ;-for PBS n i 14. tDetermined on dav 15 of infection initiated 5 X lo* trypomastig&es. ODesignates significant differences (P < 0.05).
with
each of the tissues or organs monitored. A significantly greater amount of radiolabel was found in the kidneys and small intestine of BCG treated mice
while the PBS treated mice were found to have accumulated more of the label in the liver. None of the other tissues was found to contain significantly different amounts of radiolabelled trypanosomes. DISCUSSION Under conditions used in these experiments lympho-reticular stimulation with BCG alone had no significant effect on parasitemia or longevity during the course of acute experimental Chagas’ disease in C3H(He) female mice. Caution should be expressed, however, in dismissing the possibility of BCG effectiveness under other conditions. Route of injection, dose rate, strain of BCG, and concomitant antigenic stimulation could differentially affect host immunity with resultant effects on longevity and parasitemia. BCG is considered to have several possible immunopotentiating mechanisms including enhancement of delayed type hypersensitivity (Mackaness, Lagrange & Ishibashi, 1974), “unblocking antibody” induction (Bansal & Sjogren, 1971), as well as macrophage stimulation (Nelson, 1969). In the present investigation, one or more of the components of the immune system could have been mobilized (or, perhaps, inhibited) with no measurable effect on the net immune effectiveness of BCG stimulated mice as reflected in longevity and para-
559
sitemia development. The in vivo distribution of 51Cr-labelled trypanosomes in our experimental and control mice might, indeed, indicate that BCG treatment affects the host cell-parasite interaction. The observation that BCG treated mice accumulated significantly fewer parasites in the liver than did PBS controls would suggest a greater capacity of fixed macrophage binding of parasites in the other tissues of the BCG treated mice. This explanation is supported by the increased accumulation of label in the kidneys and small intestine of the BCG stimulated mice. In the absence of demonstrated BCG induced immune enhancement in the present study, the significance of the change in in vivo distribution is not clear. It was noted in the Results that animals used for parasitemia determinations and, therefore were bled from the tail on alternate days, lived longer than animals which were merely observed for survival times. There was not a significant difference between the experimental and control groups used for parasitemia determinations or between longevities of the two PBS groups (longevity group and parasitemia group). A significant difference (P < 0.01) was observed, however, between the two BCG treated groups. Animals which were bled for parasitemia determinations lived an average of 3.6 days longer than the BCG longevity group. Since all other aspects except tail vein bleeding were identical and done from the same preparations of BCG and infective parasites, it seems that this difference must be considered real. If this difference can be attributed to increased stress with consequent cortisone release (Christian & Davis, 1964) in the bled mice then a recent study by Mackaness & Lagrange (1974) might suggest an explanation. These workers demonstrated that BCG stimulated mice when treated with cyclophosphamide, which specifically inhibits antibody formation, develop a greatly increased level of delayed type hypersensitivity over animals treated with BCG alone. The combined effects of BCG and cyclophosphamide apparently enhanced cell mediated immunity by abrogating antibody and immune complex inhibition of T cells. Among its several reported functions, cortisone is known to inhibit antibody production (Nelson, 1969) and could interact in the immune system in a fashion similar to cyclophosphamide resulting in increased delayed type hypersensitivity and greater longevity in T. cruzi infected mice. Acknowledgements-This work was supported Brown-Hazen Grant from Research Corporation.
by a
REFERENCES BANSAL S. C. & SJ~CREN H. 0.
1971. Unblocking
serum
in the polyoma system may correlate with antitumor effects of antiserum in vitro. Nature New in vitro
Biology 233: 76-77.
560
R. E. KUHN, R. T. VACGHN and G. A. HERBST
BEHBEHANI K. 1973. Developmental cycles of Trypanosoma (Schizotrypanum) cruzi (Chagas’, 1909) in mouse peritoneal macrophages in vitro. Parasitology 66: 343-353. CHRISTIAN J. J. &DAVIS D. E. 1964. Endocrines, behavior and populations. Science 146: 1550-1560. DVORAK J. A. & SCHMUN~S G. A, 1972. Trypanosoma cruzi: interaction with mouse peritoneal macrophages. Experimental Parasitology 32: 289-300. FARACI R. P. & SCHOUR L. 1974. Malignant melanoma: Specific immunity induced by B&illus CalmetteGuerin in BALB/c mice. Science 185: 68-69. GOBLE F. C. & BOYD J. L. 1962. Reticula-endothelial blockade in experimental Chagas’ disease. Journal of Parasitology 48: 233-228. KIERSZENBAUM F., KNECHT E., BUDZKO D. B. & PIZZIMENTI M. C. 1974. Phagocytosis: A defense mechanism against infection with Trypanosoma cruzi. The Journal of Immunology 112: 1839-1844. KUHN R. E., VAUGHN R. T. & IANNUZZI N. P. 1974. The
I.J.P. VOL. 5. 1975
in vivo distribution of 51Cr-labelled Trypanosoma cruzi in mice. International Journal for Parasitology 4: 585-588. LEMOND P. 1973. Protective effects of BCG and other bacteria against neoplasia in mice and hamsters. National Cancer Institute Monograph 39: 21-30. MACKANESS G. B. & LAGRANGE P. H. 1974. Restoration of cell-mediated immunity to animals blocked by a humoral response. The Journal of Experimental Medicine 140: 865-870. MACKANESS G. B., LAGRANGE P. H. & ISHIBASHI T. 1974. The modifying effect of BCG on the immunological induction of T cells. The Journal of Experimental Medicine 139: 1540-1552. NELSON D. S. 1969. Macrophages and Immunity. NorthHolland, London. TALIAFERRO W. H. & PIZZI T. 1955. Connective tissue reactions in normal and immunized mice of a reticulotrophic strain of Trypanosoma cruzi. Journal of Infectious Diseases 96: 199-228.
of Biology, Wake Forest University, Winston-Salem, (Received
17 December
North Carolina 27109, U.S.A.
1974)
Abstract-KUHN R. E., VAUGHNR. T. and HERBST G. A. 1975. The effect of BCG on the course of experimental Chagas’ disease in mice. international Jo14rnaE for ParasitoZogy 5: 557-560. Experiments were done to determine the effect of BCG treatment on longevity, development of parasitemia, and in vivo distribution of 51Cr-labelled trypanosomes in C3H(He) female mice infected with a Brazil strain of Trypanosoma cruzi. BCG sensitization of mice was accomplished by a single IV injection of 3.0 mg (wet weight) of BCG. Twenty-one days after BCG injection mice were infected with 5 x IO" blood-form trypomastigotes. Parasitemia dete~ina~i~ns were made on alternate days during the experiment while in vivo distribution of exogenously supplied 51Cr~pimastigotes was made in groups of BCG or PBS stimulated mice on day 15 of the T. cruzi infection. It was found that BCG sensitization had no effect on longevity or parasitemia development in T. cvuzi infected C3H(He) female mice, There were, however, some differences in the in vivo distribution of parasites between BCG treated and control mice. BCG stimulated mice accumulated greater numbers of radiolabelled trypanosomes in the kidneys and small intestines while PBS treated mice were found to have greater numbers of labelled parasites in the liver. Although no significant differences were observed in longevity of BCG or PBS treated mice, it was noted that BCG treated animals which were bled for parasitemia dete~inations lived significantly longer than those which were merely observed for longevity. INDEX KEY WORDS: Trypanosoma
cruzi; BCG; immunity;
THE ROLE
of the macrophage in acute Trypanosoma in mammals is not understood. Taliaferro & Pizzi (1955) found in in viva studies that macrophages can destroy these trypanosomes but that, in some cases, the parasite proliferates in macrophages and may be responsible for depletion of reticuloendothelial elements during the course of acute experimental Chagas’ disease. Behbehani (1973) later reported that, although the parasite was often killed by macrophages in vitro, in many cases amastigotes proliferated intracellularly and were capable of transfo~ing into t~pom~tigotes which could escape the macrophage to reinvade other cells. Dvorak & Schmunis (1972), on the other hand, convincingly showed that phagocytosed epimastigotes and trypomastigote forms of the parasite were killed by macrophages irz v&o and that the parasite was incapable of avoiding phagocytic destruction. In viva experiments have shown that manipulation of the reticul~ndothelial system (RES) can affect the course of experimental Chagas’ disease. Kierszenbaum, Knecht, Budzko & Pizzimenti (1974) have shown that stimulation of the RES by diethylstilbestrol results in greater longevity, reduced mortality, and decreased parsitemia in T. cruzi infected mice. cruzi
infections
557
Wr-Iabelling;
Chagas’ disease; mice.
These workers also confirmed earlier studies showing that depression of the RES results in increased severity of the disease (Goble & Boyd, 1962). The destructive capacity of macrophages may play a primary role in immune protection against many eucaryotic protozoan parasitic diseases. Enhancing macrophage activity, indeed, may be impo~ant in i~unothera~utic means of treating some of these diseases. Beneficial effects of non-specific stimulation of the immune system as reported by Kierszenbaum et al. (1974) tends to support this claim. An immune stimulant which has not been examined in a protozoan parasite system is the B&flus Camette-Guerin (BCG} which is an attenuated strain of tubercle bacilli. BCG has been extensively investigated as an immunotherapeutic agent in human and animal tumor systems and has revealed a non-specific BCG-induced regression of several tumor types (Lemond, 1973; Faraci & Schour, 1974). It has recently been demonstrated that, in addition to non-specific stimulation, BCGtreated mice may also develop an enhanced specific cytotoxicity to some experimental tumors (Faraci &. Schour, 1974). On the basis of the success of BCG stimulation 01 cell-mediated responses in some tumor systems and the effectiveness of diethystilbestrol stimulation in
558
R. E.
KUHN, R. T. VAUGHN
experimental Chagas’ disease, it seems appropriate to examine the effect of BCG on the course of T. cruzi infections in mice. MATERIALS Animals.
AND
METHODS
All mice in this study were C3H(He) females
weighing 18-20 g and were purchased from Flow Research Laboratories, Inc. (Dublin, Va.). Animals were maintained in our laboratory for two weeks prior to use in experiments and were housed 6 to a cage. Purina rat chow and water were supplied ad libitum. Infections. Mice were infected bv intraperitoneal (IP) injection of 5 x lo4 trypomastigotes (a Biazil strain) in 0.1 ml of ohosnhate buffered saline (PBS). Trvnomastigates wer; taken from stock mice‘ which &d been infected with 5 x IO* trypomastigotes 14 days earlier. Donor mice were bled from the orbital plexus into cold Alsevers’ solution and the blood subsequently centrifuged and washed three times in cold PBS. The concentration of parasites was determined by hemacytometer counting and adjusted to 5 x lo4 trypomastigotes per 0.1 ml of PBS. C3H(He) female mice (20 g) receiving this inoculum usually die on day 17 or 18. Parasitemia and longevity determinations. Separate groups of mice were used to determine the effect of BCG on parasitemia and longevity. Ten experimental and 10 control mice were used for parasitemia studies and two other groups of 10 mice were used for longevity determinations. Parasitemia in infected animals was determined by placing a 4~1 sample of tail vein blood uniformly under an 18 mm circular glass coverslip, counting the parasites in 100 fields, and converting this figure to the number of parasites per ml of blood. Parasitemia determinations were made on alternate days beginning with day 8 of the infection period. Cages were checked each morning and each afternoon for dead mice. Animals found dead in the afternoon were considered as having half-day increased longevity to animals found dead in the morning. Bacillus Calmette-Guerin (BCG). Cultures of BCG were obtained from the laboratory of Dr. Quentin Myrvik, Department of Microbiology, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, North Carolina. BCG was grown in Dubose medium (Difco Laboratories, Detroit: Michigan) at 37°C for 21 days. The bacilli were then centrifuged at 3000 rev./min for 10 min, washed with 1.0 ml PBS and weighed in tared test tubes. The cultures were resuspended-with a Broeck-tvoe tissue grinder in sufficient PBS to contain 3 mg Bdb (wet w$O.2 ml PBS. This inoculum (3 mg) was injected IV into the experimental animals. Twentyone days after BCG injection (or PBS for controls), mice were infected with 5 x lo4 trypomastigotes. In vivo distribution. The in vivo distribution of SICrlabelled culture-forms of T. cruzi was determined in BCG and PBS treated mice on day 15 of the trypanosome infection by the method of Kuhn, Vaughn & Iannuzzi (1974). Four hours after experimental mice were injected IV with 10 x lo8 labelled trypanosomes, they were bled to death from the orbital plexus and the following organs removed: liver, spleen, kidneys, heart, lungs, skeletal muscle (right upper hindleg musculature), brain, axillary and bra&al lymph nodes, and skin. The blood and
and G. A.
HERBST
I.J.P. VOL.
5, 1975
organs were then counted for radioactivity in a Nuclear Chicago well scintillation counter and the data calculated using ?he following formula: c.p.m. per specified organ x 100. total recovered c.p.m. Statistics. Data were evaluated using the Student t-test and are presented with standard deviations where applicable. RESULTS
The development of parasitemia in PBS and BCG treated mice is shown in Fig. 1. Parasitemias in the
two groups parallelled each other until day 20 after which time the BCG treated animals developed and
. / .,.;’
24 22 20
0
0 u ‘il . E
16 I6
-
I
BCG experimentols PBS controls
14
E 12 Cl S 10 g 5 I-
6 6 4
Day
of
InfectIon
FIG. I. Parasitemia values for BCG and PBS treated mice during the course of experimental Chagas’ disease.
sustained greater parasitemias. The higher parasitemia values recorded for the BCG treated mice, however, are not significantly different (P < 0.05) from the control values at any time during the course of infection. Mean longevity for the two groups of mice used for parasitemia determinations was 26.8 & 2.7 and 25.7 * 7.59 for the BCG and PBS treated groups, respectively. Table 1 contains information regarding the longevity of BCG treated and control mice. As can TABLE I-LONGEVITY
IN BCG AND PBS TREATEDC3H(He) FEMALE MICE*-i_ Mean survival
Treatment
BCG longevity group PBS longevity group BCG parasitemia group PBS parasitemia group
(davs) i,
23.2 23.4 26.8 25.7
f f f *
I.64 5.26 2.7 7.59
Ranee
20-25 15-28 20-30 18-39
* N = IO for all groups. t Longevity groups were not bled for parasitemia determinations.
be seen, the mean survival time of the BCG group, (23.2 + , _ 1.64), was not different from control
I.J.P. VOL. 5.
1975
Immunity to T~ypano~oma cruzi
animals (23.4 & 5.26). Survival times ranged from 20 to 25 days for the BCG treated mice while the control mice began dying on day 15 with 100% mortality not occurring until day 28. There were, however, some statistically significant differences in the in vivo distribution of 51-Cr-labelled trypanosomes between the two groups. Table 2 presents the data as % accumulation of label in TABLE 2--h vivo DISTRIBUTION OF TRYPANOSOMES IN BCG AND
PBS
TREATEDC3H(He)
Organ or tissue Blood Liver 0 Spleen Kidneys 0 Small intestine* Heart Lungs Skeletal muscle Brain Lymph nodes Skin
FEMALEMICE*‘~$
BCG treated 5.23 51.88 7.80 7.17 2.65 0.51 15.25 1.51 0.65 0.35 7.00
* 1.77 jr 3.27 * 1.62 i 1.39 f 0.82 i 0.44 ri_ 2.46 t 0.91 i 0.60 + 0.40 f 1.69
PBS controls 4.22 58.02 7.53 4.74 1.76 0.55 14.22 0.95 0.57 0.38 6.89
i 1.35 + 4.40 + 1.56 & 1.18 + 0.96 f 0.59 * 3.12 f 0.80 1 0.80 i 0.42 !L 2.52
*All values expressed as “/” recovered c.D.m. tFor BCG group n = 11 ;-for PBS n i 14. tDetermined on dav 15 of infection initiated 5 X lo* trypomastig&es. ODesignates significant differences (P < 0.05).
with
each of the tissues or organs monitored. A significantly greater amount of radiolabel was found in the kidneys and small intestine of BCG treated mice
while the PBS treated mice were found to have accumulated more of the label in the liver. None of the other tissues was found to contain significantly different amounts of radiolabelled trypanosomes. DISCUSSION Under conditions used in these experiments lympho-reticular stimulation with BCG alone had no significant effect on parasitemia or longevity during the course of acute experimental Chagas’ disease in C3H(He) female mice. Caution should be expressed, however, in dismissing the possibility of BCG effectiveness under other conditions. Route of injection, dose rate, strain of BCG, and concomitant antigenic stimulation could differentially affect host immunity with resultant effects on longevity and parasitemia. BCG is considered to have several possible immunopotentiating mechanisms including enhancement of delayed type hypersensitivity (Mackaness, Lagrange & Ishibashi, 1974), “unblocking antibody” induction (Bansal & Sjogren, 1971), as well as macrophage stimulation (Nelson, 1969). In the present investigation, one or more of the components of the immune system could have been mobilized (or, perhaps, inhibited) with no measurable effect on the net immune effectiveness of BCG stimulated mice as reflected in longevity and para-
559
sitemia development. The in vivo distribution of 51Cr-labelled trypanosomes in our experimental and control mice might, indeed, indicate that BCG treatment affects the host cell-parasite interaction. The observation that BCG treated mice accumulated significantly fewer parasites in the liver than did PBS controls would suggest a greater capacity of fixed macrophage binding of parasites in the other tissues of the BCG treated mice. This explanation is supported by the increased accumulation of label in the kidneys and small intestine of the BCG stimulated mice. In the absence of demonstrated BCG induced immune enhancement in the present study, the significance of the change in in vivo distribution is not clear. It was noted in the Results that animals used for parasitemia determinations and, therefore were bled from the tail on alternate days, lived longer than animals which were merely observed for survival times. There was not a significant difference between the experimental and control groups used for parasitemia determinations or between longevities of the two PBS groups (longevity group and parasitemia group). A significant difference (P < 0.01) was observed, however, between the two BCG treated groups. Animals which were bled for parasitemia determinations lived an average of 3.6 days longer than the BCG longevity group. Since all other aspects except tail vein bleeding were identical and done from the same preparations of BCG and infective parasites, it seems that this difference must be considered real. If this difference can be attributed to increased stress with consequent cortisone release (Christian & Davis, 1964) in the bled mice then a recent study by Mackaness & Lagrange (1974) might suggest an explanation. These workers demonstrated that BCG stimulated mice when treated with cyclophosphamide, which specifically inhibits antibody formation, develop a greatly increased level of delayed type hypersensitivity over animals treated with BCG alone. The combined effects of BCG and cyclophosphamide apparently enhanced cell mediated immunity by abrogating antibody and immune complex inhibition of T cells. Among its several reported functions, cortisone is known to inhibit antibody production (Nelson, 1969) and could interact in the immune system in a fashion similar to cyclophosphamide resulting in increased delayed type hypersensitivity and greater longevity in T. cruzi infected mice. Acknowledgements-This work was supported Brown-Hazen Grant from Research Corporation.
by a
REFERENCES BANSAL S. C. & SJ~CREN H. 0.
1971. Unblocking
serum
in the polyoma system may correlate with antitumor effects of antiserum in vitro. Nature New in vitro
Biology 233: 76-77.
560
R. E. KUHN, R. T. VACGHN and G. A. HERBST
BEHBEHANI K. 1973. Developmental cycles of Trypanosoma (Schizotrypanum) cruzi (Chagas’, 1909) in mouse peritoneal macrophages in vitro. Parasitology 66: 343-353. CHRISTIAN J. J. &DAVIS D. E. 1964. Endocrines, behavior and populations. Science 146: 1550-1560. DVORAK J. A. & SCHMUN~S G. A, 1972. Trypanosoma cruzi: interaction with mouse peritoneal macrophages. Experimental Parasitology 32: 289-300. FARACI R. P. & SCHOUR L. 1974. Malignant melanoma: Specific immunity induced by B&illus CalmetteGuerin in BALB/c mice. Science 185: 68-69. GOBLE F. C. & BOYD J. L. 1962. Reticula-endothelial blockade in experimental Chagas’ disease. Journal of Parasitology 48: 233-228. KIERSZENBAUM F., KNECHT E., BUDZKO D. B. & PIZZIMENTI M. C. 1974. Phagocytosis: A defense mechanism against infection with Trypanosoma cruzi. The Journal of Immunology 112: 1839-1844. KUHN R. E., VAUGHN R. T. & IANNUZZI N. P. 1974. The
I.J.P. VOL. 5. 1975
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