Edwin C. Rowland

Isotype determination of anti-Trypanosoma cruzi antibody in murine Chagas' disease.

Isotypic analysis of anti-parasite humoral responses of C57B1/6 and C3H (He) mice surviving acute Trypanosoma cruzi infection showed that both mouse strains demonstrate IgG1, IgG2a, IgG2b, and IgM enzyme-linked immunosorbent assay titers from days 21 to 300 of infection. Using the western blot technique to determine the antigen specificity of the isotypic responses, 100-day infected C3H mice showed strong IgG1, IgG2a, and IgG2b responses to many antigens, whereas C57B1/6 mice showed weak responses to fewer antigens. Isotype western blots showed that reactivity to the T. cruzi antigen of 75-77 kDa is present in the humoral response of day 21-infected mice that will survive and missing in those that will not survive. In general, surviving immunized C3H mice respond with IgG1, IgG2a, and IgG2b reactions to the 75-77-kDa and other antigens, whereas resistant B6 mice concentrate their anti-T. cruzi response in the IgG2b isotype to the 75-77-kDa antigen. Perhaps induction of ineffective antibody responses to nonprotective antigens is beneficial to the parasite and detrimental to the host.

Corpus Christi Strain-Induced Protection to Trypanosoma cruzi Infection in C3H(He) Mice: Transfer of Resistance to Brazil Strain Challenge with Lymphocytes

Treatment of susceptible C3H(He) mice with 10(7) live Corpus Christi strain culture-derived Trypanosoma cruzi provided protection against a subsequent Brazil strain challenge. This protection was indicated by a greater than 10-fold decrease in parasitemia and an increase in longevity (including survival) in many groups. The Corpus Christi organisms were unable to establish an apparent infection, but viability is an important element in the treatment in that freeze-thawed, non-viable preparations of the Corpus Christi strain were unable to provide protection. Adoptive transfer of resistance was achieved with spleen cells from Corpus Christi-treated, Brazil-infected mice which had recovered from the acute phase of infection. The T cell-depleted population of these spleen cells was able to transfer resistance whereas the T cell-enriched population was not protective. Passive transfer of serum from Corpus Christi-treated and Brazil-infected mice provided a temporary decrease in parasitemia in infected mice. The results presented herein suggest that Corpus Christi-induced protection to virulent T. cruzi challenge is mediated by antibody mechanisms.

A ROLE FOR PROTEASE ACTIVITY AND HOST-CELL PERMEABILITY DURING THE PROCESS OF TRYPANOSOMA CRUZI EGRESS FROM INFECTED CELLS

The mechanism by which Trypanosoma cruzi egresses from infected cells at the end of the intracellular replication cycle is not understood. This study explored the role of T. cruzi–derived proteases and host-cell membrane permeability during the parasites egress process. Treatment with a fluoromethyl ketone, known to inhibit the parasites major protease, significantly reduced parasite egress. In addition, in the late stages of intracellular infection, cells infected with T. cruzi showed increased permeability as evidenced by dye exclusion tests. Furthermore, parasites could be antibody stained inside host cells without chemical permeabilization of the plasma membrane. These results suggest that in advanced stages of the intracellular cycle of T. cruzi, the host cells lose membrane integrity. Previous studies in our laboratory have found that antibodies present in sera of mice chronically infected with T. cruzi (antiegressin) bind the surface of infected cells and reduce parasite egress. In agreement with these reports, western blot analysis showed that several proteins in infected cell membrane extracts reacted with antibodies from infected mouse serum. The findings reported herein might have implications in the process of T. cruzi egress, as well as in the mechanism of action of antiegressin.

CORPUS CHRISTI STRAIN-INDUCED PROTECTION TO TRYPANOSOMA CRUZI INFECTION IN C3H(He) MICE: EFFECTIVE DOSE, TIME, ROUTE, AND NUMBER OF VACCINATIONS

The study of the parameters affecting Corpus Christi strain-induced protection in C3H(He) mice against Brazil strain T. cruzi infection is reported herein. A dose of 10(7) Corpus Christi epimastigotes was found to be the most effective dose for protection. Vaccination of mice 5 days to 11 wk prior to infection was determined to be the optimal time interval for protection. The subcutaneous route for vaccination and infection provides the most effective protection to experimental animals. Multiple inoculations with Corpus Christi, whether live or freeze thawed, increased the protective effect only slightly. The Corpus Christi strain of T. cruzi has proved to be quite suitable in providing protection to highly susceptible C3H(He) mice against an infection with the virulent Brazil strain of T. cruzi.

Inhibition of In Vitro Intracellular Growth of Trypanosoma cruzi by Dicationic Compounds

Dicationic compounds, which are derivatives of pentamidine, are being developed for use as antiprotozoal drugs. These compounds bind to the minor groove of DNA and are thought to inhibit DNA-dependent enzymes and thereby prevent cellular replication by protozoans. The objective of this study was to test the ability of a group of these compounds to inhibit the intracellular and extracellular reproduction of Trypanosoma cruzi in vitro. At present, there are few drugs in use capable of inhibiting the intracellular stages of this parasite, and therefore compounds with this ability would be of value. Cultures of mouse fibroblasts were infected and treated with doses of dicationic compounds, and the numbers of parasites released at the end of the 5- to 7-day growth cycle were determined. Five of the compounds tested were found to be effective at inhibiting T. cruzi growth at doses that were not toxic to the host cells. The compound found most effective (DB709) inhibited parasite release at the low concentration of 0.8 ng/ml, justifying further study. These results suggest that dicationic compounds may have potential as chemotherapy against T. cruzi infection.

Infection Characteristics of an Ecuadorian Trypanosoma cruzi Strain with Reduced Virulence

A human isolate of Trypanosoma cruzi obtained from Guayaquil, Ecuador (Guayas strain) was examined for its infectivity of the resistant C57Bl/6 (B6) and the susceptible C3H (He) mouse strains and compared to infection with the known virulent Brazil strain. C3H mice were capable of surviving acute Guayas infection, whereas the Brazil infection was fatal for this mouse strain. Both C3H and B6 mice showed a greatly reduced (over 10-fold) parasitemia during Guayas infection compared to Brazil infection. Histologic examination of heart tissue from Guayas-infected B6 and C3H mice indicates little inflammation, unlike what is typically seen in B6 mice chronically infected with the Brazil strain. There appears to be no remarkable difference in the anti-parasite antibody responses (as measured by ELISA and western blot) in mice infected 100 days with Guayas or Brazil parasites. Western blot analysis of the anti-heart response indicates no response during Guayas infection to a 43-kDa heart tissue glycoprotein that is a target of antibodies from B6 mice infected with Brazil strain. The Guayas strain, therefore, provides an infection that generates a low parasitemia and strong anti-parasite responses in the absence of specific anti-heart autoimmunity and obvious myocarditis. In vitro infection characteristics of these 2 parasite strains were studied in cultures of macrophages, myocytes, and fibroblasts by microscopic examination of stained slide cultures. In both short-term (24 hr) and long-term (15 day) experiments, Brazil strain infection was shown to have a greater infection rate with a higher number of parasites per cell than Guayas infection for all host cell types.(ABSTRACT TRUNCATED AT 250 WORDS)

Trypanosoma cruzi antigen-specific antibody response in immunized mice during acute and chronic infection.

Preimmunization with living attenuated Corpus Christi strain Trypanosoma cruzi renders otherwise susceptible C3H(He) mice resistant and enhances the resistance of C57BL/6J mice to infection with the virulent Brazil Strain. Antibody titers determined by enzyme-linked immunosorbent assay were higher in immunized and subsequently infected mice compared to those immunized alone. Elevated levels of anti-T. cruzi antibodies were maintained through day 300 of infection. Sera from groups of normal, immunized, immunized and infected, and infected mice show reactivities to parasite antigens that range from 25 to 160 kDa by western blot analysis. Electrophoretically separated antigen extracts from Corpus Christi and Brazil strains of parasites have essentially identical banding patterns when reacted with mouse sera from the above-mentioned groups. However, antibodies that bind parasite antigens of 75 and 77 kDa are found in the sera of mice that can survive infection but not in those that will succumb to infection. These antibodies are present 21 days after infection in preimmunized C3H and B/6 mice and on day 42 of infection in the genetically resistant, unimmunized, B/6 mice. A reaction to a 65-68-kDa triplet band is found similarly in immunized and infected C3H mice, but resistant B/6 mice were found to be unresponsive to this glycoprotein during infection. In addition, antibodies were elicited to a 31-kDa parasite antigen in all mice infected with the Brazil strain regardless of preimmunization status. These data show that antibodies with specificity for the 75- and 77-kDa antigens correlate with protection against virulent parasites and may confer this protection.

ANTIEGRESSIN ACTS LATE IN THE INTRACELLULAR GROWTH CYCLE OF TRYPANOSOMA CRUZI TO INHIBIT PARASITE EGRESS

Trypanosoma cruzi is the causative agent of Chagas disease, which is characterized by acute and chronic phases. During the former, parasitemia rises dramatically, then decreases significantly during the chronic phase. Immune mechanisms responsible for the parasitemia reduction have not been thoroughly elucidated. The goal of the present study was to further characterize the immune response during chronic infection. Previously, we described antiegressin, an antibody in sera from chronically infected mice. The in vitro presence of antiegressin inhibits parasite egress from infected host cells. Antiegressin appears by day 14 of an in vivo infection and is maintained through at least day 280 postinfection. The in vitro functional activity of antiegressin is initiated late in the 4–6 days intracellular growth cycle of T. cruzi; antiegressin may be added at day 4, inhibiting parasite release at day 5. Immunocytochemical staining using antineuraminidase demonstrates the presence of mature parasites inside host BALB/c fibroblasts grown in the presence of antiegressin. These results demonstrate the ability of antiegressin to inhibit emergence of developmentally mature trypomastigotes from infected host cells late in their intracellular growth cycle. We believe this antibody plays an important and novel role in achieving the low-parasitemia characteristic of chronic Chagas disease.

INHIBITION OF TRYPANOSOMA CRUZI EGRESS FROM INFECTED FIBROBLASTS IS MEDIATED BY CD4+ AND μ+ IMMUNE CELLS

Trypanosoma cruzi, the causative agent of Chagas disease, is able to reproduce intracellularly in many host cell types while in the mammalian host. Although cellular immunity is known to be important in resistance to infection, the ability of immune cells to interfere with the completion of the intracellular growth cycle of T. cruzi has not been described. Using a tissue culture system to study the parasite growth cycle, we have found that spleen cells from infected mice are able to decrease the number of parasites released from infected fibroblasts. Spleen cells from mice infected for as few as 14 days and as long as 300 days display this inhibitory ability. Parasite egress from infected cells is inhibited by factor(s) released by immune cells during coculture with infected fibroblasts. Immune cell depletion studies indicate that the inhibitory activity requires the presence of both CD4+ T cells and μ+ B cells. These results suggest a direct ability of immune cells to somehow interfere with the completion of the intracellular cycle, and this ability may play a role in control of this parasite.

Parasite antigen-induced IFNγ and IL-4 production by cells from pathopermissive and pathoresistant strains of mice infected with Trypanosoma cruzi

The cardiac pathology associated with infection by Trypanosoma cruzi in mice has been suggested to be partially dependent upon cytokine responses. The pathoresistant B10.D2 mice, which display little infection-induced myocarditis, and the pathopermissive DBA/2 mice, which show significant cardiac damage, were compared for their in vitro interferon (IFN)-gamma and interleukin (IL)-4 production. Concanavalin A-stimulated spleen cells from infected B10.D2 mice produce a greater amount of IFN-gamma than DBA/2 mice, whereas the IL-4 production is only slightly greater in the B10.D2 mice than the DBA/2 mice. Parasite antigen stimulation of spleen cells from these mice results in a clearly greater IFN gamma production by the B10.D2 and a higher IL-4 level for the DBA/2 mice. The data presented suggest a relationship between an enhanced TH1-type response and decreased chronic cardiac pathogenesis, whereas a lower level of TH1 activity may play a role in cardiac involvement.