Alterations of the microcirculatory network during the clearance of epimastigote forms of Trypanosoma cruzi: an intravital microscopic study.

The distribution of epimastigote forms of Trypanosoma cruzi in the microcirculatory network and the vessel alterations were observed using an intravital microscopy technique. Immediately after intravenous inoculation of 2 x 10(6) epimastigote suspension into normal mice, parasites were seen as circulating clumps, and their retention at some sites of the endothelium of venules and capillaries was observed. Injection of 2 x 10(7) and 2 x 10(8) parasite suspensions induced, respectively, intermittent or total stasis of venules and capillaries, probably via obstruction by clumping. The mobility of epimastigotes in the clumps indicates that parasites were alive in the lumen of vessels. The retention of clumps in the capillaries, although intense, could only be observed when labeled parasites were inoculated. These results suggest that the rapid clearance of epimastigote forms of T. cruzi from the blood circulation of mice may be due to the retention of parasites to the endothelium of venules and capillaries that, in turn, may facilitate phagocytosis. This may be a mechanism by which mice are able to eliminate epimastigote forms from the circulation. These findings are consistent with our previous observations showing that epimastigotes are not lysed by complement activation but are phagocytosed and destroyed by a distinct population of blood cells.

How are antibodies involved in the protective mechanism of susceptible mice infected with T. cruzi?

Host resistance to Trypanosoma cruzi is dependent on both natural and acquired immune responses. During the acute phase of the infection the presence of IFN-gamma, TNF-alpha, IL-12 and GM-CSF has been closely associated with resistance, whereas TGF-ss and IL-10 have been associated with susceptibility. Several investigators have demonstrated that antibodies are responsible for the survival of susceptible animals in the initial phase of infection and for the maintenance of low levels of parasitemia in the chronic phase. However, how this occurs is not yet understood. Our results and other data in the literature support the hypothesis that the protective role of antibodies in the acute phase of infection is dependent mostly on their ability to induce removal of bloodstream trypomastigotes from the circulation in addition to other concomitant cell-mediated events.

Changes of anti-Trypanosoma cruzi antibodies after gamma-irradiation of mice in the chronic phase of the infection.

The effect of sublethal whole body irradiation (800 rads) on the level and biological activities of antibodies in mice chronically infected with the CL strain of Trypanosoma cruzi was studied. Irradiated mice died, although a high parasitemia did not always preceded death. Before and after irradiation, a constant level of antibodies was detected by enzyme-linked immunosorbent assay and complement mediated lysis, but after irradiation the level of clearance antibodies was decreased. These results suggest that clearance antibodies are important in the control of the chronic phase of the infection.

How are antibodies involved in the protective mechanism of susceptible mice infected with T. cruzi?

Host resistance to Trypanosoma cruzi is dependent on both natural and acquired immune responses. During the acute phase of the infection the presence of IFN-gama, TNF-alpha, IL-12 and GM-CSF has been closely associated with resistance, whereas TGF-beta and IL-10 have been associated with susceptibility. Several investigators have demonstrated that antibodies are responsible for the survival of susceptible animals in the initial phase of infection and for the maintenance of low levels of parasitemia in the chronic phase. However, how this occurs is not yet understood. Our results and other data in the literature support the hypothesis that the protective role of antibodies in the acute phase of infection is dependent mostly on their ability to induce removal of bloodstream trypomastigotes from the circulation in addition to other concomitant cell-mediated events.

Biological activity of chronic phase antibodies eluted from sensitized Trypanosoma cruzi trypomastigotes.

Chronic infection with Trypanosoma cruzi induces high levels of antibodies that have lytic and clearance activities on bloodstream trypomastigote forms. These two activities were tested with antibodies eluted from parasites sensitized with serum obtained from mice in the chronic phase of infection. Parasites submitted to treatment for antibody elution were also tested. Our results show that antibodies eluted from the parasites are very efficient to induce lysis but unable to induce clearance. In addition, we observed that after being submitted to treatment for antibody elution the parasites still presented a slower but significant clearance and a high lytic activity. These results allow us to suggest that clearance inducing antibodies are mostly high affinity antibodies that could not be eluted from the parasites in our experimental conditions.

One fate of bloodstream trypomastigote forms of Trypanosoma cruzi after immune clearance: an ultrastructural study.

The fate of bloodstream forms of Trypanosoma cruzi in tissues of mice was studied after immune elimination from circulation. Observations using transmission electron microscopy showed platelet thrombi occluding small vessels in the lung, liver, and spleen, and phagocytosed parasites in different stages of destruction within macrophages, neutrophils, and eosinophils. It is suggested that no particular cell population is a potential effector, but that different cells act in concert to destroy the parasites. The mechanism of this destruction might be related to intra- and extracellular mechanisms with trypanolytic activity.

One fate of epimastigote forms of Trypanosoma cruzi injected in mice. An ultrastructural study.

Recently, we suggested that epimastigote forms of Trypanosoma cruzi are cleared from circulation of mice by a mechanism independent of lysis and that platelets play an important role in this process. These observations prompted us to look at the fate of epimastigotes in the lung, liver, and spleen of mice injected intravenously with these parasite forms. Using transmission electron microscopy, we observed clumps of epimastigotes and platelets in direct contact with phagocytes in the lumen of capillaries. However, the platelets and parasites were probably separated before phagocytosis because only parasites were found inside the phagocytes. Indeed, most of the phagocytes, although containing epimastigotes in different stages of disintegration, contained no platelets. The removal of parasites from platelets was probably mediated by phagocytes through a mechanism similar to the removal of bacteria from the surface of erythrocytes in humans. These observations suggest that the nonvirulence of T. cruzi epimastigotes in mice is not due to lysis but probably to the inability of these parasite forms to escape destruction by the phagocytes.

Clearance-inducing antibodies are responsible for protection against the acute phase of Trypanosoma cruzi infection in mice

A study was conducted on mice infected with strains Y and CL of Trypanosoma cruzi. The ability of anti-Y and anti-CL sera to induce complement-mediated lysis, immune clearance and protection against the acute phase of the infection was studied using homologous anti-Y or anti-CL serum tested with the Y or CL strain, or heterologous anti-Y serum tested with the CL strain or anti-CL serum tested with the Y strain. Complement-mediated lysis was induced by both homologous and heterologous antisera but protection was afforded only by homologous antisera. Immune clearance was induced by homologous but not by heterologous antisera. Antisera with high clearance ability were able to confer protection whereas antisera with high lytic ability were not. These results show a high correlation between the antibody ability to induce clearance and to confer protection and suggest that clearance rather than lysis is responsible for protection against the acute phase of the infection. The mechanisms of antibody protection against the acute phase of the infection is discussed.

Clearance-inducing antibodies are responsible for protection against the acute phase of Trypanosoma cruzi infection in mice.

A study was conducted on mice infected with strains Y and CL of Trypanosoma cruzi. The ability of anti-Y and anti-CL sera to induce complement-mediated lysis, immune clearance and protection against the acute phase of the infection was studied using homologous anti-Y or anti-CL serum tested with the Y or CL strain, or heterologous anti-Y serum tested with the CL strain or anti-CL serum tested with the Y strain. Complement-mediated lysis was induced by both homologous and heterologous antisera but protection was afforded only by homologous antisera. Immune clearance was induced by homologous but not by heterologous antisera. Antisera with high clearance ability were able to confer protection whereas antisera with high lytic ability were not. These results show a high correlation between the antibody ability to induce clearance and to confer protection and suggest that clearance rather than lysis is responsible for protection against the acute phase of the infection. The mechanism of antibody protection against the acute phase of the infection is discussed.

Specificity and role of anti-Trypanosoma cruzi clearance antibodies.

Two strains of Trypanosoma cruzi (Y and CL) were used to study the specificity and role of anti-T. cruzi clearance antibodies. Clearance antibodies were only induced after immunization with living blood-stream trypomastigotes (Btrys) but not with dead parasites. Btrys of either strain were readily cleared from the circulation after passive immunization with anti-Y or anti-CL serum provided that the homologous strain was used. CL or Y Btrys sensitized in vitro with the homologous or heterologous antiserum and transferred to normal mice were cleared from the circulation only when the homologous antiserum was used. Clearance antibodies were removed from serum by absorption with the homologous but not with the heterologous strain. Clearance antibodies were removed from serum by absorption with living Btrys but not with fixed parasites. These results suggest that: a) the parasite epitopes involved in the clearance are peculiar to each strain, b) the clearance antibodies are specific to these epitopes, and c) a proper conformation of the parasite antigens is required for the induction and effector activity of the clearance antibodies.

Specificity and role of anti-Trypanosoma cruzi clearance antibodies

Two strains of Trypanosoma Cruzi (Y and CL) were used to study the specificity and role of anti-T. cruzi clearance antibodies. Clearance antibodies were only induced after immunization with living blood-stream trypomastigotes (Btrys) but not with dead parasites. Btrys of either strain were readily cleared from the circulation after passive immunization with anti-Y or anti-CL scrum provided that the homologous strain was used. CL or Y Btrys sensitized in vitro with the homologous or heterologous antiserum and transferred to normal mice were cleared from the circulation only when the homologous antiserum was used. Clearance antibodies were removed from serum by absorption with the homologous but not with the heterologous strain. Clearance antibodies were removed from serum by absorption with living Btrys but not with fixed parasites. These results suggest that: a) the parasite epitopes involved in the clearance are peculiar to each strain, b) the clearance antibodies are specific to these epitopes, and c) a proper conformation of the parasite antigens is required for the induction and effector activity of the clearance antibodies.

Role of platelets and complement in the clearance of epimastigote forms of Trypanosoma cruzi.

1. Trypanosoma cruzi epimastigote forms are very rapidly removed from the circulation of normal and C5-deficient mice. Depletion of C3 by cobra venom factor results in a significant delay in parasite clearance. 2. During parasite clearance there is a significant decrease in the number of circulating platelets and parasite clearance is considerably delayed in thrombocytopenic animals. 3. In vitro incubation of epimastigote forms with normal mouse serum leads to the formation of parasite clumps provided that platelets are present. Inactivation of factor B or depletion of C3 prevents this phenomenon. 4. When epimastigotes are incubated with normal mouse serum they absorb one or more factors required for their aggregation with platelets. 5. It is suggested that in mice T. cruzi epimastigote forms are removed from circulation by the alternative pathway of complement activation and that both C3 and platelets are required for parasite clearance.

Role of platelets and complement in the clearance of epimastigote forms of Trypanosoma cruzy

1. Trypanosoma cruzi epimastigote forms are very rapidly removed from the circulation of normal and C5-deficient mice. Depletion of C3 by cobra venom factor results in a significant delay in parasite clearance. 2. During parasite clearance there is a significant decrease in the number of circulating platelets and parasite clearance is considerably delayed in thrombocytopenic animals. 3. In vitro incubation of epimastigote forms with normal mouse serum leads to the formation of parasite clumps provided that platelets are present. Innactivation of factor B or depletion of C3 prevents this phenomenon. 4. When epimastigotes are incubated with normal mouse serum they absorb one or more factors required for their aggregation with platelets. 5. It is suggested that in mice T. cruzi epimastigote forms are removed from circulation by the alternative pathway of complement activation and that both C3 and platelets are required for parasite clearance

Role of platelets in the in vivo removal of T. cruzi from circulation.

The possible role of platelets in the clearance of Trypanosoma cruzi was studied in vivo in A/sn female mice. Platelet depletion achieved by anti-platelet IgG antibodies induced a significant, though not total, reduction in the rate of removal of T. cruzi bloodstream trypomastigotes (BTRYS) from the circulation. Furthermore, during removal of T. cruzi BTRYS from the circulation of normal mice there was a simultaneous decrease in the number of platelets. These results suggest that platelets play a role in the in vivo mechanism of defense against T. cruzi infection.

Role of platelets in the in vivo removal of T. cruzi from circulation

The possible role of platelets in the clearance of Trypanosoma cruzi was studied in vivo in A/sn female mice. Platelet depletion achieved by anti-platelet IgG antibodies induced a significant, though not total, reduction in the rate of removal of T. cruzi bloodstream trypomastigotes (BTRYS) from the circulation. Furthermore, during removal of T. cruzi BTRYS from the circulation of normal mice there was a simultaneous decrease in the number of platelets. These results suggest that platelets play a role in the in vivo mechanism of defense against T. cruzi infection

Role of the mononuclear phagocytic system in the immune and nonspecific clearance of Trypanosoma cruzi bloodstream trypomastigotes

The removal of T. cruzi bloodstream trypomastigotes (BTRYS) from the circulation is mediated mostly by the mononuclear phagocytic system (MPS). In the present study we investigated the the nonspecific and the immune clearance of BTRYS in groups of 4 mice whose MPS activity was either enhanced by BCG treatment or depressed by silica treatment. Treatment with BCG resulted in a significant increase in the nonspecific clearance of both carbon particles (100% after 6 min) and BTRYS (60% after 5 min) 28 days after BCG treatment but there was no change in the immune clearance of the parasites. Pretreatment of the animals with silica induced a significant reduction of the colloidal carbon clearance (80% less than control 15 min later) but did not alter the nonspecific or the immune clearance of BTRYS. We conclude that the removal of the opsonized parasites from the circulation is due to a mechanism different from that of the nonspecific clearance

In-vitro lysis of sensitized Trypanosoma cruzi by platelets: role of C3b receptors.

Incubation of Trypanosoma cruzi bloodstream trypomastigotes (Btrys) with C5-deficient blood in the presence of anti-T. cruzi immune mouse serum (IMS) or its IgG fraction resulted in an immediate formation of small clumps in which one could easily see platelets adhered to the parasites. After 4 h of incubation most of the clumps had disappeared and the number of the parasites was considerably reduced. Twenty-four hours later there were only a few remaining parasites. This same sequence of events was also observed when the parasites were incubated with isolated platelets in presence of IMS or its IgG fraction. When the parasites were incubated with plasma in the presence of IMS or its IgG fraction but in the absence of platelets there was strong agglutination of the parasites but no reduction in their number. Incubation of the parasites with platelets and IMS or its IgG fraction in C3-depleted plasma prevented adherence of the platelets to the parasites and their subsequent lysis. It is concluded that platelets are able to induce in-vitro lysis of the Btrys and that this phenomenon is dependent on the platelet' receptor for C3b.

The effect of C3 depletion on the clearance of Trypanosoma cruzi induced by IgG antibodies.

Passive transfer of homologous immune serum or IgG anti-Trypanosoma cruzi antibodies to normal mice containing circulating T. cruzi bloodstream trypomastigotes (Btrys) induces a very fast clearance of the parasites. Previous work from this laboratory has shown that F(ab')2 fragments obtained from IgG anti-T. cruzi antibodies retain the ability to induce lysis of the Btrys but are unable to induce clearance of the parasites. This suggests that the clearance is dependent on the Fc region. The removal of the Btrys may then be effected by attachment of the antibodies to the parasites and removal of the opsonized parasites by the mononuclear phagocytic system. Attachment of the opsonized parasites to macrophages may be effected either through the Fc receptor that binds specifically to the heavy chain of IgG or through C3b fragments after cleavage of C3 by C3-convertases. In order to find out the possible role of C3b in this phenomenon the clearance of Btrys was determined in mice depleted of C3 by previous treatment with cobra venom factor. The results of these experiments showed that depletion of C3 completely abolished the immune clearance induced by anti-T. cruzi antibodies. It is suggested that C3 is required for the clearance of Btrys from circulation.

Role of the mononuclear phagocytic system in the immune and nonspecific clearance of Trypanosoma cruzi bloodstream trypomastigotes.

1. The removal of T. cruzi bloodstream trypomastigotes (BTRYS) from the circulation is mediated mostly by the mononuclear phagocytic system (MPS). In the present study we investigated the nonspecific and the immune clearance of BTRYS in groups of 4 mice whose MPS activity was either enhanced by BCG treatment or depressed by silica treatment. 2. Treatment with BCG resulted in a significant increase in the nonspecific clearance of both carbon particles (100% after 6 min) and BTRYS (60% after 5 min) 28 days after BCG treatment but there was no change in the immune clearance of the parasites. 3. Pretreatment of the animals with silica induced a significant reduction of the colloidal carbon clearance (80% less than control 15 min later) but did not alter the nonspecific or the immune clearance of BTRYS. 4. We conclude that the removal of the opsonized parasites from the circulation is due to a mechanism different from that of the nonspecific clearance.

Role of the antibody Fc in the immune clearance of Trypanosoma cruzi.

Passive transfer of immune serum obtained from mice chronically infected with Trypanosoma cruzi to mice containing circulating bloodstream trypomastigotes induces a very fast clearance of the parasites. Comparison of trypomastigotes clearance in normocomplementemic and C5-deficient mice showed no difference. IgG fraction obtained from immune serum was very efficient at inducing complement-mediated lysis and immune clearance of bloodstream trypomastigotes, whereas its Fc-missing F (ab') 2 fragments, although able to induce lysis, were unable to induce clearance. It is suggested that the immune clearance of bloodstream trypomastigotes is dependent on the antibody Fc region and that complement-mediated lysis is not a prerequisite for elimination of the parasites from circulation.