Biological action of pyrazolopyrimidine derivatives against Trypanosoma cruzi. Studies in vitro and in vivo

The capacity of 54 different pyrazolo(3,4-d) or (4,3-d)pyrimidine derivatives to inhibit Trypanosoma cruzi epimastigote and trypomastigote multiplication, and for some of them its chemotherapeutic activity, was evaluated. Six pyrazolo(3,4-d)pyrimidines showed inhibitory activity against epimastigote forms, 4-aminopyrazolo(3,4-d)pyrimidine being the most active, 5-fold more so than 4-hydroxypyrazolo(3,4-d)-pyrimidine. Neither compound was active against freshly isolated trypomastigotes, suggesting biochemical differences between culture and bloodstream forms of T. cruzi. On both epimastigote and trypomastigote forms, 7-amino-3-beta-D-ribofuranosylpyrazolo-(4,3-d)pyrimidine (FoA) was about 2-fold more active than 7-hydroxy-3-beta-D-ribofuranosylpyrazolo-(4,3-d)pyrimidine (FoB); however, when tested on T. cruzi-infected mice, only FoB exhibited significant chemotherapeutic activity. Previous results suggest that, except for FoB and FoA: (a) pyrazolopyrimidine insensitivity is trypomastigote-specific and (b) drug-insensitivity is lost when trypomastigotes transform into epimastigotes and vice versa.     

Changes in fibroblast-derived trypomastigotes of Trypanosoma cruzi during long-term culture.

Fibroblast-derived trypomastigotes (FDTs) of Trypanosoma cruzi that had been in culture for extended periods of time were found to differ in their ability to proliferate in culture when compared to blood-form trypomastigotes (BFTs) and FDTs that had been recently established from blood-forms. "Old" FDTs transform into amastigotes/spheromastigotes and epimastigotes and readily incorporate [3H]thymidine in medium alone or in the presence of mouse spleen cells, whereas "new" FDTs and BFTs did not incorporate [3H]thymidine although they did transform in culture. These differences should be considered when FDTs are used for physiologic and immunologic studies of T. cruzi.     

Altered nucleoside transporters in mammalian cells selected for resistance to the physiological effects of inhibitors of nucleoside transport

From a mutagenized population of wild type S49 T lymphoma cells, clones were generated that were resistant to the physiological effects of the potent inhibitor of nucleoside transport, 4-nitrobenzyl-6-thioinosine (NBMPR). These cells were selected for their ability to survive in semisolid medium containing 0.5 mM hypoxanthine, 0.4 microM methotrexate, 30 microM thymidine, 30 microM deoxycytidine, in the presence of 30 microM NBMPR. NBMPR protected wild type cells from the effects of a spectrum of cytotoxic nucleosides, whereas two mutant clones, KAB1 and KAB5, were still sensitive to nucleoside-mediated cytotoxicity in the presence of NBMPR. Comparisons of the abilities of wild type cells and mutant cells to incorporate exogenous nucleoside to the corresponding nucleoside triphosphate indicated that the KAB1 and KAB5 mutant cells were refractory to normal inhibition by NBMPR. Moreover, rapid transport studies indicated that mutant cells, unlike wild type parental cells, had acquired a substantial NBMPR-insensitive nucleoside transport component. Binding studies with [3H]NBMPR indicated that KAB5 cells were 70-75% deficient in the number of NBMPR binding sites, whereas KAB1 cells possessed a wild type complement of NBMPR binding sites. These data suggest that the NBMPR binding site in wild type S49 cells is genetically distinguishable from the nucleoside carrier site.     

Pentamidine transport and sensitivity in brucei-group trypanosomes.

Sensitivity to pentamidine of bloodstream forms and culture forms of Trypanosoma brucei brucei, strains of this subspecies, and strains of T. brucei rhodesiense characteristically differs in vitro. Analyses of transport parameters for pentamidine uptake in these organisms show differences that correspond with drug sensitivity. Long slender bloodstream forms of T. b. brucei have a high affinity for the drug and high rates of uptake at indicated by Km and Vmax values for [3H]pentamidine transport. Although pentamidine and stilbamidine resistance is associated with dyskinetoplasty, this condition does not itself confer resistance to pentamidine nor does it affect pentamidine transport. However, drug-resistant strains show lower rates for pentamidine transport as does T. b. rhodesiense, which is characteristically less sensitive to the drug. Of all the forms and strains studied, procyclic trypomastigotes were least sensitive to pentamidine and had a remarkable ability to exclude the drug.     

Genetic analysis of 2',3'-dideoxycytidine incorporation into cultured human T lymphoblasts

In order to analyze the cellular determinants that mediate the action of 2',3'-dideoxycytidine, the growth inhibitory and cytotoxic effects and the metabolism of the dideoxynucleoside were examined in wild type human CEM T lymphoblasts and in mutant populations of CEM cells that were genetically deficient in either nucleoside transport or deoxycytidine kinase activity. Whereas 2',3'-dideoxycytidine at a concentration of 5 microM inhibited growth of the wild type CEM parental strain by 50%, two nucleoside transport-deficient clones were 4-fold resistant to the pyrimidine analog. The deoxycytidine kinase-deficient cell line was virtually completely resistant to growth inhibition by the dideoxynucleoside at a concentration of 1024 microM. An 80% diminished rate of 2',3'-[5,6-3H]dideoxycytidine influx into the two nucleoside transport-deficient lines could account for their resistance to the dideoxynucleoside, while the resistance of the deoxycytidine kinase-deficient cells to 2',3'-dideoxycytidine toxicity could be explained by a virtually complete failure to incorporate 2',3'-[5,6-3H]dideoxycytidine in situ. Two potent inhibitors of mammalian nucleoside transport, 4-nitrobenzylthioinosine and dipyridamole, mimicked the effects of a genetic deficiency in nucleoside transport with respect to 2',3'-dideoxycytidine toxicity and incorporation. These data indicate that the intracellular metabolism of 2',3'-dideoxycytidine in CEM cells is initiated by the nucleoside transport system and the cellular deoxycytidine kinase activity.     

Invasion of MDCK epithelial cells with altered expression of Rho GTPases by Trypanosoma cruzi amastigotes and metacyclic trypomastigotes of strains from the two major phylogenetic lineages

In order to invade mammalian cells, Trypanosoma cruzi infective forms cause distinct rearrangements of membrane and host cell cytoskeletal components. Rho GTPases have been shown to regulate three separate signal transduction pathways, linking plasma membrane receptors to the assembly of distinct actin filament structures. Here, we examined the role of Rho GTPases on the interaction between different T. cruzi infective forms of strains from the two major phylogenetic lineages with nonpolarized MDCK cells transfected with different Rho GTPase constructs. We compared the infectivity of amastigotes isolated from infected cells (intracellular amastigotes) with forms generated from the axenic differentiation of trypomastigotes (extracellular amastigotes), and also with metacyclic trypomastigotes. No detectable effect of GTPase expression was observed on metacyclic trypomastigote invasion and parasites of Y and CL (T. cruzi II) strains invaded to similar degrees all MDCK transfectants, and were more infective than either G or Tulahuen (T. cruzi I) strains. Intracellular amastigotes were complement sensitive and showed very low infectivity towards the different transfectants regardless of the parasite strain. Complement-resistant T. cruzi I extracellular amastigotes, especially of the G strain, were more infective than T. cruzi II parasites, particularly for the Rac1V12 constitutively active GTPase transfectant. The fact that in Rac1N17 dominant-negative cells, the invasion of G strain extracellular amastigotes was specifically inhibited suggested an important role for Rac1 in this process.     

Dipyridamole-insensitive nucleoside transport in mutant murine T lymphoma cells

From a mutagenized population of S49 murine T lymphoma cells, a mutant cell line, JPA4, was selected that expressed an altered nucleoside transport capability. JPA4 cells transported low concentrations of purine nucleosides and uridine more rapidly than the parental S49 cell line. The transport of these nucleosides by mutant cells was insensitive to inhibition by either dipyridamole (DPA) or 4-nitrobenzylthioinosine (NBMPR), two potent inhibitors of nucleoside transport in mammalian cells. Kinetic analyses revealed that the apparent Km values for the transport of uridine, adenosine, and inosine were 3-4-fold lower in JPA4 cells compared to wild type cells. In contrast, the transport of both thymidine and cytidine by JPA4 cells was similar to that of parental cells, and transport of these pyrimidine nucleosides remained sensitive to inhibition by both NBMPR and DPA. Furthermore, thymidine was a 10-12-fold weaker inhibitor of inosine transport in JPA4 cells than in wild type cells. Thus, JPA4 cells appeared to express two types of nucleoside transport activities; a novel (mutant) type that was insensitive to inhibition by DPA and NBMPR and transported purine nucleosides and uridine, and a parental type that retained sensitivity to inhibitors and transported cytidine and thymidine. The phenotype of the JPA4 cell line suggests that the sensitivity of mammalian nucleoside transporters to both NBMPR and DPA can be genetically uncoupled from its ability to transport certain nucleoside substrates and that the determinants on the nucleoside transporter that interact with each nucleoside are not necessarily identical.     

Enrichment of right-side-out Trypanosoma cruzi plasma membrane vesicles

A simple method to prepare a high yield of Trypanosoma cruzi plasma membrane vesicles (PMV) from epimastigotes and metacyclic trypomastigotes is described. The method may be applicable to other protozoa. Solid-phase immunoassay to bind surface T. cruzi epitopes showed that this preparation was enriched with 80-82% PMV and that most of these were right-side out (81-92%). The method was based on the extraction of extrinsic proteins and subpellicular tubules with mild high and low ionic strength buffers without detergents (pH 7.4) and on the differential centrifugation of PMV based on their specific density (1.049 g/ml, 4 degrees C). Transmission electron microscopy of PMV pellets showed a heterogeneous population of vesicles without other significant cytoskeletal contaminants. T. cruzi PMV were also enriched with an ouabain- and oligomycin-insensitive magnesium-ATPase and contained an adenylyl cyclase, preserved for at least 3 months at -70 degrees C in storage buffer. Measurements of the [14C]-dextran and the 3H2O space indicated that T. cruzi PMV were not sealed, explaining why Lubrol PX and NaF failed to stimulate the adenylyl cyclase activity further and why T. cruzi PMV were unable to concentrate 86Rb in flow dialysis assays. No detectable DNA and RNA was found. The preparation was not capable of removing 51Cr or [3H]glucosamine from live L6 myoblast surfaces in physiologic conditions and acid phosphatase was extracted by this method. The contaminating fraction (18-20% by immunoassay) consisted of endoplasmic reticulum membranes with NADH oxidase activity and of kinetoplast membranes with cytochrome c oxidase and oligomycin sensitive magnesium-ATPase activity. The biologically active T. cruzi PMV retained the ability of living forms to trigger the alternate pathway of complement by releasing the Bb activation fragment from human Factor B.     

Heterogeneity of [3H]Dipyridamole Binding to CNS Membranes: Correlation with [3H]Nitrobenzylthioinosine Binding and [3H]Uridine Influx Studies

The relationship between the nucleoside transport system and the nitrobenzylthioinosine-sensitive and -resistant [3H]dipyridamole binding sites was examined by comparing the characteristics of [3H]dipyridamole binding with those of [3H]nitrobenzylthioinosine binding and [3H]-uridine influx in rabbit and guinea pig cerebral cortical synaptosomes. Two distinct high-affinity synaptosomal membrane-associated [3H]dipyridamole binding sites, with different sensitivities to inhibition by nitrobenzylthioinosine, were characterized in the presence of 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS, 0.01%) to prevent [3H]dipyridamole binding to glass tubes and filters. The nitrobenzylthioinosine-resistant [3H]-dipyridamole binding sites represented a greater proportion of the total membrane sites in guinea pig than in rabbit (40 vs. 10% based on inhibition studies). In rabbit, nitrobenzylthioinosine-sensitive [3H]dipyridamole binding (KD = 1.4 +/- 0.2 nM) and [3H]nitrobenzylthioinosine binding (KD = 0.30 +/- 0.01 nM) appeared to involve the same membrane site associated with the nitrobenzylthioinosine-sensitive nucleoside transporter. By mass law analysis, [3H]-dipyridamole binding in guinea pig could be resolved into two components based on sensitivity to inhibition by 1 microM nitrobenzylthioinosine. The nitrobenzylthioinosine-resistant [3H]dipyridamole binding sites were relatively insensitive to inhibition by all of the nucleoside transport substrates and inhibitors tested, with the exception of dipyridamole itself. In guinea pig synaptosomes, 100 microM dilazep blocked nitrobenzylthioinosine-resistant [3H]uridine transport completely but inhibited the nitrobenzylthioinosine-resistant [3H]dipyridamole binding component by only 20%. Furthermore, a greater percentage of the [3H]dipyridamole binding was nitrobenzylthioinosine resistant in guinea pig compared with rabbit, yet both species had a similar percentage of nitrobenzylthioinosine-resistant [3H]uridine transport.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleoside transport. Photoaffinity labelling of high-affinity nitrobenzylthioinosine binding sites in rat and guinea pig lung

Binding of the potent nucleoside transport inhibitor [3H]nitrobenzylthioinosine to rat and guinea pig lung membranes was investigated. Reversible high-affinity binding was found in both species (apparent KD approximately 0.3nM). Binding was inhibited by nitrobenzylthioguanosine, adenosine and uridine. Dipyridamole was also an effective inhibitor of [3H]nitrobenzylthioinosine binding to guinea pig membranes. In contrast, rat membranes were relatively insensitive to dipyridamole. Exposure of site-bound [3H]nitrobenzylthioinosine to high intensity U.V. light resulted in the photoaffinity labelling of lung proteins with apparent molecular weights similar to that of the human erythrocyte nucleoside transporter (45,000-65,000).     

Exacerbation of experimental Trypanosoma cruzi infection in mice by concomitant malaria.

The effect of malaria on the chronic phase of Chagas' disease was investigated in mice. The animals were given Plasmodium bergheri-infected red blood cells 2 to 12 months after their initial inoculation with trypomastigotes of 3 different strains of Trypanosoma cruzi (Y. CL and Gilmar). in all the experiments carried out with one of the strains (CL), a somewhat variable but always considerable percentage of mice (average 39%) relapsed in to the acute phase of Chagas' disease. This relapse was characterized by a significant increase in the number of circulating trypomastigotes. Recrudescence was observed also with a 2nd strain of T. cruzi (Gilmar), which is similar in many aspects to the CL strain, e.g. the morphology of blood stages, curved of parasitemia and susceptibility to antibodies in vitro. In mice whose chronic phase was induced by trypomastigotes of the Y strain, malaria infections did not induce a typical acute phas with high parasitemia by T. cruzi. Bloodstream forms of Y parasites differ from those of CL and Gilmar strains morphologically as well as immunologically, i.e. only the Y strain is easily agglutinated and partly inactivated by specific immune serum. In light of this and other known characteristics of the strains used in the present work, the author speculates on mechanisms which allow malaria infections selectively to suppress acquired host resistance to certain strains of T. cruzi.     

Genetic analysis of the 6-thiobenzylpurine binding site of the nucleoside transporter in mouse lymphoblasts

From a mutagenized population of wild-type S49 T lymphoblasts, cells were selected for their ability to survive in semisolid medium containing 0.5 mM hypoxanthine, 0.4 microM methotrexate, 30 microM thymidine, 30 microM deoxycytidine, and 30 microM p-nitrobenzyl-6-thioinosine (NBMPR), a potent inhibitor of nucleoside transport. Unlike wild-type parental cells, two mutant clones, KAB1 and KAB5, were still sensitive to nucleoside-mediated cytotoxicity in the presence of NBMPR. Comparisons of the abilities of wild-type cells, KAB1, and KAB5 cells to incorporate exogenous nucleoside to the corresponding nucleoside triphosphate indicated that nucleoside incorporation was much less sensitive to inhibition by NBMPR in the mutant cells. Rapid transport studies indicated that the mutant cell lines, unlike the wild-type parent, had acquired an NBMPR-insensitive nucleoside transport component which was similar to the NBMPR-sensitive wild-type transporter with respect to affinities for nucleosides and sensitivities toward N-ethylmaleimide and dipyridamole. Binding studies with [3H]NBMPR indicated that KAB5 cells were 70-75% deficient in the number of NBMPR binding sites, whereas KAB1 cells possessed a wild-type complement of NBMPR binding sites with wild-type binding characteristics. These data suggest that the NBMPR binding site in wild-type S49 cells is genetically distinguishable from the nucleoside carrier site and that the former may be a regulatory site.     

Pentamidine Transport and Sensitivity in brucei-Group Trypanosomes*

SYNOPSIS. Sensitivity to pentamidine of bloodstream forms and culture forms of Trypanosoma brucei brucei, strains of this subspecies, and strains of T. brucei rhodesiense characteristically differs in vitro. Analyses of transport parameters for pentamidine uptake in these organisms show differences that correspond with drug sensitivity. Long slender bloodstream forms of T. b. brucei have a high affinity for the drug and high rates of uptake as indicated by K_m and V_max values for [³H]pentamidine transport. Although pentamidine and stilbamidine resistance is associated with dyskinetoplasty. this condition does not itself confer resistance to pentamidine nor does it affect pentamidine transport. However, drug-resistant strains show lower rates for pentamidine transport as does T. b. rhodesiense, which is characteristically less sensitive to the drug. Of all the forms and strains studied, procyclic trypomastigotes were least sensitive to pentamidine and had a remarkable ability to exclude the drug.     

Sodium-dependent and inhibitor-insensitive uptake of adenosine by mouse peritoneal exudate cells

[8-3H]Adenosine uptake in mouse peritoneal exudate cells, harvested following i.p. challenge with Complete Freund's Adjuvant from BALB/c mice, was found to be insensitive to common nucleoside transport inhibitors such as dilazep or 6-[(4-nitrobenzyl)mercapto]purine ribonucleoside and to require sodium ion, being inactive when sodium was replaced by lithium or potassium. These findings also applied to the adherent (macrophages) and nonadherent (polymorphonuclear cells) cell fractions prepared from the peritoneal cell mixture. Uptake was inhibited by several nucleosides including deoxyadenosine, inosine, uridine, thymidine and, to a lesser extent, by the adenosine analog tubercidin, while adenine, fructose, glucose and ribose were without effect. Uptake [8-3H]adenosine was fully matched by rapid intracellular phosphorylation to AMP, ADP and ATP. Inosine was a substrate for the transporter, but tubercidin was not. The system clearly is distinct from carrier-mediated, nonconcentrative transport and has similarities to concentrative, sodium-dependent nucleoside transporters described in other cell types.     

Biological characterization of Trypanosoma cruzi strains from different zymodemes and schizodemes.

The development in C3H mice of thirteen strains of Trypanosoma cruzi belonging to different zymodemes and schizodemes was studied. Host mortality, virulence, histiotropism, parasitemia and polymorphism of the parasites were recorded. The strains were grouped into: a) high virulence--causing 100% mortality and characterized by predominance of very broad trypomastigotes in the bloodstream at the end of infection; b) medium virulence--causing no mortality and with a predominance of broad trypomastigotes; c) low virulence--causing no mortality with blood forms not described due to the very low parasitemia. During 18 months maintenance the parasitemia curves were kept constant for all strains except one. A direct correlation between either zymodeme or schizodeme and experimental biological properties of T. cruzi strains was not found. However, the parasitemia was subpatent and patent for strains from zymodeme C and the others respectively. Furthermore the high virulence seems to be related to one of two schizodemes found within zymodeme B strains. All strains presenting patent parasitemia independent of shizodeme and zymodeme showed a myotropism towards heart and skeletal muscle with variable inflammatory intensity. The present study confirmed the heterogeneity found by isoenzyme and k-DNA patterns among the strains of T. cruzi isolated from chagasic patients in Bambuí, Minas Gerais State, Brasil.     

Thymidine incorporation in nucleoside transport-deficient lymphoma cells

Nucleoside transport deficiency in mammalian cells is associated with an inability to transport most nucleosides, growth resistance to a spectrum of cytotoxic nucleosides, and a loss of binding sites for 4-nitrobenzylthioinosine (NBMPR), a potent inhibitor of nucleoside transport. The nucleoside transport-deficient S49 T lymphoma cell line, AE1, however, was almost as capable of incorporating thymidine into TTP as the wild type parent provided thymidine was administered at a sufficiently high concentration. Consequently, AE1 cells were just as sensitive as wild type cells to the toxicity of high thymidine concentrations. In contrast, AE1 cells were highly resistant to almost all other cytotoxic nucleosides including the thymidine analogs, 5-bromodeoxyuridine and 5-fluoro-2'-deoxyuridine 5'-monophosphate. Despite having demonstrable ability to accumulate TTP, AE1 cells were unable to grow on hypoxanthine-amethopterin-thymidine (HAT)-containing medium. This was due to their inability to accumulate sufficient TTP from the low concentrations of thymidine present in HAT medium. AE1 cells possessed an incomplete thymidine transport deficiency, the extent of which was concentration dependent. The residual capacity for thymidine transport present in AE1 cells was insensitive to inhibition by 4-nitrobenzylthioinosine and could account both for their inability to grow on HAT medium and their sensitivity to cytotoxic concentrations of thymidine. Another nucleoside transport-deficient cell line, FURD-80-3-6, was similar to the AE1 cell line in its growth phenotype and NBMPR-binding site deficiency but differed in its decreased growth sensitivity to thymidine. That nucleoside transport deficiencies may vary in their completeness for different nucleosides has significance for the mechanism by which a single transporter can recognize a wide variety of nucleosides.     

Inhibition of Protein Synthesis and Amino Acid Transport by Crystal Violet in Trypanosoma cruzi

ABSTRACT. [³⁵S]methionine incorporation into proteins of either T. cruzi epimastigotes or trypomastigotes was drastically inhibited by low concentrations of crystal violet in a dose-dependent manner. This inhibition was not due to ATP depletion since cellular ATP levels did not change significantly after incubation of epimastigotes with 50 μM crystal violet for similar periods of time, and was unaffected by changes in the extracellular free calcium concentration. Although crystal violet was able to inhibit protein synthesis in a cell-free system from T. cruzi epimastigotes, half maximal inhibition was at 1 mM, a concentration three orders of magnitude higher than those that inhibited protein synthesis in intact cells. On the other hand, crystal violet was able to inhibit total [³⁵S]methionine uptake at similar concentrations to those that inhibited protein synthesis while addition of increasing concentrations of cold methionine to the incubation medium protected the cells against crystal violet inhibition. Crystal violet also inhibited total [³H]proline uptake thus indicating that it has a general inhibitory effect upon the transport of amino acids, and not specifically upon methionine. These results indicate that inhibition of protein synthesis by crystal violet is probably due to inhibition of amino acid uptake.     

Isolation and characterization of a mutant of L1210 murine leukemia deficient in nitrobenzylthioinosine-insensitive nucleoside transport

L1210 mouse leukemia cells exhibit two distinct types of nucleoside transport activity that have similar kinetic properties and substrate specificity, but differ markedly in their sensitivity to the inhibitor nitrobenzylthioinosine (NBMPR) (Belt, J. A. (1983) Mol. Pharmacol. 24, 479-484). It is not known whether these two transport activities are mediated by a single protein or by separate and distinct nucleoside transport proteins. We have isolated a mutant from the L1210 cell line that has lost the NBMPR-insensitive component of nucleoside transport, but retains NBMPR-sensitive transport. In the parental cell line 20-40% of the nucleoside transport activity is insensitive to 1 microM NBMPR. In the mutant, however, uridine and thymidine transport are almost completely inhibited by NBMPR. Consistent with the loss of NBMPR-insensitive transport, the mutant cells can be protected from the toxic effects of several nucleoside analogs by NBMPR. In contrast, the toxicity of the same analogs in the wild type cells is not significantly affected by NBMPR, presumably due to uptake of the nucleosides via the NBMPR-insensitive transporter. On the other hand, NBMPR-sensitive transport in the mutant appears to be unaltered. The mutant is not resistant to cytotoxic nucleosides in the absence of NBMPR and the cells retain the wild type complement of high affinity binding sites for NBMPR. Furthermore, the affinity of the binding site for the inhibitor is similar to that of parental L1210 cells. These results suggest that NBMPR-sensitive and NBMPR-insensitive nucleoside transport in L1210 cells are mediated by genetically distinct proteins. To our knowledge this is the first report of a mutant deficient in NBMPR-insensitive nucleoside transport.     

The Attraction of Trypanosoma cruzi to Vertebrate Cells In Vitro

SYNOPSIS. A video technic is described that permits a quantification of the degree of attraction of Trypanosoma cruzi trypomastigotes to vertebrate cells in vitro. Bovine embryo skeletal muscle cells (BESM), HeLa cells and Vero cells all attract a myotropic strain of T. cruzi trypomastigotes. BESM cells, however, are 2-fold more attractive to trypomastigotes than HeLa cells and 10-fold more attractive than Vero cells. Heat-inactivation of BESM cells abolishes their ability to respire and also to attract T. cruzi trypomastigotes. As there is no difference in the endogenous oxygen consumption between BESM, HeLa, and Vero cells, it is unlikely that differences in the attraction of trypomastigotes to the 3 cell types are due to variations in the magnitude of pO₂ or pCO₂ gradients in the milieu around the cells.