Growth and Differentiation of Trypanosoma cruzi Cultivated with a Triatoma infestans Embryo Cell Line*

SYNOPSIS. Trypanosoma cruzi strain Peru was cultivated in the presence of an established cell line of Triatoma infestans embryo cells (TI-32). Bloodstream trypomastigotes differentiated into amastigote-like cells (first differentiation phase) which multiplied to form large clusters of cells. Because of their clustering nature, a new term, “staphylomastigotes,” has been proposed for this stage. After 10 days of cultivation, 90% of the staphylomastigotes underwent differentiation (2nd differentiation phase) to trypomastigotes (?98%) or epimastigotes (?2%). Bloodstream trypomastigotes cultivated without TI-32 cells underwent the first, but not the 2nd differentiation phase, although occasional epimastigotes were seen (< 1%). The evidence presented suggests that TI-32 cells produce a labile factor(s) important not only for initiation of the 2nd differentiation phase but also for maintaining the parasites in the trypomastigote stage. The pH of the culture medium was not the initiating factor for the 2nd differentiation phase. Infectivity studies indicated that staphylomastigotes were as infective as bloodstream trypomastigotes, but that metacyclic trypomastigotes isolated from culture after the 2nd differentiation phase were slightly more infective than bloodstream forms. Electromicrographs of styphylomastigotes do not provide any evidence of exchange of genetic material between cells.     

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.     

In vitro production of metacyclic forms of Trypanosoma musculi and their infectivity in CBA mice

Bloodstream forms of Trypanosoma musculi, cultured in Schneider's drosophila medium at room temperature, multiply and differentiate through a series of developmental stages into infective metacyclic trypomastigotes in 10 days. Oral inoculation with these culture forms into CBA mice produced a parasitemia similar to that produced by intraperitoneal infection with bloodstream forms except for a three-day longer prepatent period. Attempts to induce parasitemia with bloodstream forms given orally were unsuccessful.     

Exacerbation of Experimental Trypanosoma cruzi Infection in Mice by Concomitant Malaria*

SYNOPSIS. The effect of malaria on the chronic phase of Chagas’disease was investigated in mice. The animals were given Plasmodium berghei-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, curve 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 phase 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.     

Features of host cell invasion by different infective forms of Trypanosoma cruzi

Through its life cycle from the insect vector to mammalian hosts Trypanosoma cruzi has developed clever strategies to reach the intracellular milieu where it grows sheltered from the hosts' immune system. We have been interested in several aspects of in vitro interactions of different infective forms of the parasite with cultured mammalian cells. We have observed that not only the classically infective trypomastigotes but also amastigotes, originated from the extracellular differentiation of trypomastigotes, can infect cultured cells. Interestingly, the process of invasion of different parasite infective forms is remarkably distinct and also highly dependent on the host cell type.     

In Vitro Production of Metacyclic Forms of Trypanosoma musculi and Their Infectivity in CBA Mice1

ABSTRACT. Bloodstream forms of Trypanosoma musculi, cultured in Schneider's drosophila medium at room temperature, multiply and differentiate through a series of developmental stages into infective metacyclic trypomastigotes in 10 days. Oral inoculation with these culture forms into CBA mice produced a parasitemia similar to that produced by intraperitoneal infection with bloodstream forms except for a three-day longer prepatent period. Attempts to induce parasitemia with bloodstream forms given orally were unsuccessful.     

Lectin receptors in Trypanosoma cruzi. An N-acetyl-D-glucosamine-containing surface glycoprotein specific for the trypomastigote stage

We have investigated the interaction of three lectins, differing in their sugar specificities, with the surface of the three differentiation stages of Trypanosoma cruzi. The Scatchard constants for each lectin and parasite stage imply that differentiation of T. cruzi is accompanied by changes in the cell surface saccharides. Trypomastigotes obtained from two different sources do not differ appreciably as to the number and affinity of binding sites for the three lectins employed, suggesting a similar cell-surface saccharide composition. These conclusions are reinforced by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the 131I-labeled surface glycoproteins, following isolation by affinity chromatography. The surface membrane of trypomastigotes, the infective stage to T. cruzi for mammalian cells, possesses a specific glycoprotein of apparent Mr 85000 (Tc-85) which is absent from the other two stages and can be isolated by affinity chromatography on wheat germ agglutinin-Sepharose columns. This glycoprotein also binds to concanavalin A, but not to Lens culinaris lectin. The binding of Tc-85 to wheat germ agglutinin is unaffected by treatment of either the isolated glycoprotein or intact living trypomastigotes with neuraminidase. Since N-acetyl-D-glucosamine inhibits internalization of trypomastigotes by cultured mammalian cells, it is suggested that Tc-85 might be involved in adhesion and/or interiorization of T. cruzi into mammalian cells, possibly via recognition of an ubiquitous host-cell surface N-acetyl-D-glucosamine-specific receptor activity.     

A reappraisal of "T-independent" antigens. II. Studies on single, hapten-specific B cells from neonatal CBA/H or CBA/N mice fail to support classification into TI-1 and TI-2 categories

Spleen cells from adult CBA/H or CBA/N mice, or from neonatal CBA/H mice, were fractionated on thin layers of fluorescein (FLU)-gelatin to yield FLU-specific B lymphocytes. A single cell, or small numbers ranging from 1 to 10, were cultured in 10-microliter microcultures together with various antigens and mitogens. The results were compared with those of bulk culture or limiting dilution cultures supported by thymus filler cells. B cell growth and differentiation-promoting conditioned media (BGDA) were added to some cultures. The CBA/N results gave no support to the commonly used classification of T cell-independent (TI) antigens into TI-1 and TI-2 categories. A typical supposed TI-1 antigen, FLU-LPS, strongly stimulated normal adult single FLU-specific B cells to proliferate and form antibody, but virtually failed to trigger CBA/N B cells of comparable antigen-binding avidity. The same was true of LPS or LPS plus dextran sulfate acting as mitogens. The allegedly TI-2 antigen FLU-Ficoll, although still triggering comparatively poor responses, was actually marginally more active than FLU-LPS. FLU-Brucella abortus (FLU-BA) + BGDA gave the best results with single CBA/N B cells, but still induced only 1.27% of cells to develop into antibody-forming clones vs 12.2% with CBA/H cells. The results obtained with single neonatal B cells also lent no support to the distinction between TI-1 and TI-2. Both "TI-1" and "TI-2" stimuli caused adequate proliferation, one "TI-2" antigen stimulating 23.2% of the cells. None of the antigens caused good antibody formation, however, probably because multivalent antigens can deliver signals impeding the differentiation of immature B cells. It is therefore suggested that the classification of TI-1 antigens into two subcategories be abandoned, at least for the time being.     

Trypanosoma rhodesiense Bloodstream Trypomastigote and Culture Procyclic Cell Surface Carbohydrates1, 2, 3

Bloodstream trypomastigote and culture procyclic (insect midgut) forms of a cloned T. rhodesiense variant (WRATat 1) were tested for agglutination with the lectins concanavalin A (Con A), phytohemagglutinin P (PP), soybean agglutinin (SBA), fucose binding protein (FBP), wheat germ agglutinin (WGA), and castor bean lectin (RCA). Fluorescence-microscopic localization of lectin binding to both formalin-fixed trypomastigotes and red cells was determined with fluorescein isothiocyanate (FITC)-conjugated Con A, SBA, FBP, WGA, RCA, PNA (peanut agglutinin), DBA (Dolichos bifloris), and UEA (Ulex europaeus) lectins. Electron microscopic localization of lectin binding sites on bloodstream trypomastigotes was accomplished by the Con A-horseradish peroxidase-diaminobenzidine (HRP-DAB) technique, and by a Con A-biotin/avidin-ferritin method. Trypomastigotes, isolated by centrifugation or filtration through DEAE-cellulose or thawed after cryopreservation, were agglutinated by the lectins Con A and PP with agglutination strength scored as Con A < PP. No agglutination was observed in control preparations or with the lectins WGA, FBA or SBA. Red cells were agglutinated by all the lectins tested. Formalin-fixed bloodstream trypomastigotes bound FITC-Con A and FITC-RCA but not FITC-WGA, -SBA, -PNA, -UEA or -DBA lectins. All FITC-labeled lectins bound to red cells. Con A receptors, visualized by Con A-HRP-DAB and Con A-biotin/avidin-ferritin techniques, were distributed uniformly on T. rhodesiense bloodstream forms. No lectin receptors were visualized on control preparations. Culture procyclics lacked a cell surface coat and were agglutinated by Con A and WGA but not RCA, SBA, PP and FBP. Procyclics were not agglutinated by lectins in the presence of competing sugar at 0.25 M. The expression of lectin binding cell surface saccharides of T. rhodesiense WRATat 1 is related to the parasite stage. Sugars resembling α-D-mannose are on the surface of bloodstream trypomastigotes and culture procyclics; n-acetyl-D-galactosamine and D-galactose residues are on bloodstream forms; and n-acetyl-D-glucosamine-like sugars are on procyclic stages.     

Differential apoptosis-like cell death in amastigote and trypomastigote forms from Trypanosoma cruzi-infected heart cells in vitro

Apoptosis, type-I of programmed cell death (PCD-I), is not restricted to multicellular organisms since many apoptotic features have been described in different trypanosomatids, including Trypanosoma cruzi. Our present aim was to monitor, by different morphological markers, the occurrence of apoptosis-like death in amastigotes and trypomastigotes of T.cruzi (Y strain) during the infection of heart culture cells. We documented the differential occurrence of PCD-I in amastigotes and trypomastigotes, with distinct death rates noticed between these two parasite-distinct forms. Fluorescence microscopy and flow cytometry analysis using different hall markers of apoptosis (phosphatidylserine exposure, collapse of mitochondrial membrane potential and DNA fragmentation) showed that amastigotes present higher levels of apoptosis-like cell death as compared to trypomastigotes. It is possible that the higher levels of PCD-I in these highly multiplicative forms may contribute to the control of the parasite burden within the host cells. On the other hand, the apoptosis-like occurrence in the infective but non-proliferative stage of the parasite (trypomastigotes) may play a role in parasite evasion mechanisms as suggested for other parasites.     

Biological characterization of Trypanosoma cruzi zymodemes: in vitro differentiation of epimastigotes and infectivity of culture metacyclic trypomastigotes to mice

Thirty-one Trypanosoma cruzi isolates from Chile, Peru, and Bolivia were studied in their capacity to differentiate in vitro from epimastigotes to metacyclic trypomastigotes on TAU-3AAG medium. Zymodeme 1 parasites displayed the best level of differentiation, which ranges from 60 to 90% depending on the isolate. Zymodeme 2 parasites exhibited highly heterogenous differentiation rates. This differentiation method permits the obtention of large amounts of metacyclic trypomastigotes from zymodeme 1 parasites. Metacyclic trypomastigotes obtained in vitro were infective to nude Balb/c hybrid mice. Zymodeme 1 parasites produced high parasitemias in this murine model; in contrast, zymodeme 2 parasites displayed lower parasitemias. Of a total of 27 T. cruzi isolates, 20 proved to be infective to mice, 12 gave enough parasites for further studies, and 8 of these were used for biological characterization. Results are compared with the infective clone Dm28 and Tulahuén strains maintained since 1954 in mice.     

A conserved domain of the gp85/trans-sialidase family activates host cell extracellular signal-regulated kinase and facilitates Trypanosoma cruzi infection

Chagas' disease is a chronic, debilitating and incapacitating illness, caused by the protozoan parasite Trypanosoma cruzi when infective trypomastigotes invade host cells. Although the mechanism of trypomastigotes interaction with mammalian cells has been intensively studied, a final and integrated picture of the signal transduction mechanisms involved still remains to be elucidated. Our group has previously shown that the conserved FLY domain (VTVXNVFLYNR), present in all members of the gp85/trans-sialidase glycoprotein family coating the surface of trypomastigotes, binds to cytokeratin 18 (CK18) on the surface of LLC-MK(2) epithelial cells, and significantly increases parasite entry into mammalian cells. Now it is reported that FLY, present on the surface of trypomastigotes or on latex beads binds to CK18, promotes dephosphorylation and reorganization of CK18 and activation of the ERK1/2 signaling cascade culminating in an increase of approximately 9-fold in the number of parasites/cell. Inhibition of ERK1/2 phosphorylation completely blocks the adhesion of FLY to cells and blocks by 57% the host cell infection by T. cruzi. Taken together our results indicate that the conserved FLY domain is an important tool that trypomastigotes have evolved to specific exploit the host cell machinery and guarantee a successful infection.     

Inhibition of HSP90 in Trypanosoma cruzi induces a stress response but no stage differentiation

The 90-kDa heat shock proteins (HSP90) are important in the regulation of numerous intracellular processes in eukaryotic cells. In particular, HSP90 has been shown to be involved in the control of the cellular differentiation of the protozoan parasite Leishmania donovani. We investigated the role of HSP90 in the related parasite Trypanosoma cruzi by inhibiting its function using geldanamycin (GA). GA induced a dose-dependent increase in heat shock protein levels and a dose-dependent arrest of proliferation. Epimastigotes were arrested in G₁ phase of the cell cycle, but no stage differentiation occurred. Blood form trypomastigotes showed conversion towards spheromastigote-like forms when they were cultivated with GA, but differentiation into epimastigotes was permanently blocked. We conclude that, similar to leishmanial HSP90, functional HSP90 is essential for cell division in T. cruzi and serves as a feedback inhibitor in the cellular stress response. In contrast to L. donovani cells, however, T. cruzi cells treated with GA do not begin to differentiate into relevant life cycle stages.     

Changes in polypeptide expression following Trypanosoma cruzi differentiation from trypomastigotes to amastigotes.

Changes in the dynamic of expression of polypeptides following the differentiation from infective trypomastigotes to multiplicative amastigote forms of Trypanosoma cruzi were mapped by two-dimensional gel electrophoresis and quantitatively analyzed by laser densitometry. Following the differentiation from trypomastigotes to amastigotes the expression of the polypeptides 212, 183, 176, 149, 50-55, 43, 39, 34 and 28 kDa is turned off in multiplicative amastigotes, whereas the expression of the polypeptides 80, 66 (p.Is. 6.75-7.50), 42 and 38 kDa is turned on. After complete differentiation from trypomastigotes to amastigotes the expression of the polypeptides 43, 42, 33, 32, 29 and 23 kDa is up-regulated in amastigotes, whereas the expression of the acidic polypeptides 66 (p.Is. 6.27-6.64), 45-48 and 41-43 kDa is down-regulated.     

Continuous Cultivation of Trypanosoma theileri at 37 C in Bovine Cell Culture*

SYNOPSIS. Trypanosoma theileri was cultivated at 37 C in bovine bone marrow cell culture through 50 consecutive subcultures. Medium 199, supplemented with Bacto-peptone, vitamin B₁₂, and fetal bovine serum, was utilized both for primary and continuous cultivation. The number of trypanosomes produced in culture averaged 8 × 10⁶ (1–26 × 10⁶) trypanosomes/ml. In each subculture the organisms divided as epimastigotes and transformed into trypomastigotes; a round form was observed during the stationary and declining phase of growth. Gradual changes such as increased generation time, size reduction, and decreased trypomastigote production were observed as subculturing progressed. Cultured trypanosomes were infective for the bovine through the 48th serial transfer and could be cultivated at 26 C.     

Trypanosoma cruzi: isolation of cloned strains and characterization of their infectivity

Cloning of Trypanosoma cruzi strains Y, CL and Colombiana was achieved by plating on solid medium. Clones were obtained either from culture epimastigotes or from bloodstream trypomastigotes. In both cases the efficiency of plating was almost 100%. Clones from culture epimastigotes did not infect the albino mouse, while clones from bloodstream trypomastigotes remained infective even after several passages in a blood-agar/BHI biphasic medium, in which the amastigote-like forms prevail.     

Early preantral mouse follicle in vitro maturation: oocyte growth, meiotic maturation and granulosa-cell proliferation

Mechanically isolated early preantral mouse follicles were cultured singly for 16 d and fully grown oocytes were obtained from these follicles. We then compared in vitro and in vivo follicle growth by trypsinising the follicles and counting their cell numbers in a Neubauer-counting chamber and recording the diameter and meiotic status of oocytes under an inverted microscope. As long as the granulosa cells were within the basal membrane, proliferation was slow. From Day 6, when granulosa cells had broken through the basal membrane, the proliferation rate progressed up to Day 10 and decreased thereafter to approximately 12,000 cells per culture droplet. Incorporation of BrdU revealed that proliferating cells were evenly distributed throughout the follicle until antrum formation. As granulosa cell differentiation progressed, proliferation of mural-granulosa cells ceased, while cells around the oocytes continued dividing. Oocyte diameter increased discontinuously in relation to follicle remodelling. During the first growth phase, diameters increased from 56.5 (+/- 4.4 microns) to 67 (+/- 4.1 microns) until the onset of antral-like cavity formation. The last growth phase started after Day 10, and by Day 14 oocyte diameters were not significantly different from those of 26-d-old in vivo control oocytes. The potential to resume meiosis after mechanical removal of granulosa cells was first reached on Day 8; thereafter, removal of the corona showed that all oocytes cultured with FSH remained arrested at the GV stage up to Day 16. After Day 8, approximately 70% of all oocytes underwent GVBD as a result of granulosa-cell removal, but only 23% of these reached MII after 24 h. The in vivo controls reached a comparable GVBD rate (66%) when the granulosa was removed, but most of the oocytes (82%) underwent first polar body extrusion 24 h later. These results suggest that although oocyte diameters after IVM are not different from those of the controls, culture conditions are not yet adequate to support complete meiotic maturation.     

Trypanosoma rhodesiense bloodstream trypomastigote and culture procyclic cell surface carbohydrates

Bloodstream trypomastigote and culture procyclic (insect midgut) forms of a cloned T. rhodesiense variant (WRAT at 1) were tested for agglutination with the lectins concanavalin A (Con A), phytohemagglutinin P (PP), soybean agglutinin (SBA), fucose binding protein (FBP), wheat germ agglutinin (WGA), and castor bean lectin (RCA). Fluorescence-microscopic localization of lectin binding to both formalin-fixed trypomastigotes and red cells was determined with fluorescein isothiocyanate (FITC)-conjugated Con A, SBA, FBP, WGA, RCA, PNA (peanut agglutinin), DBA (Dolichos bifloris), and UEA (Ulex europaeus) lectins. Electron microscopic localization of lectin binding sites on bloodstream trypomastigotes was accomplished by the Con A-horseradish peroxidase-diamino-benzidine (HRP-DAB) technique, and by a Con A-biotin/avidin-ferritin method. Trypomastigotes, isolated by centrifugation or filtration through DEAE-cellulose or thawed after cryopreservation, were agglutinated by the lectins Con A and PP with agglutination strength scored as Con A greater than PP. No agglutination was observed in control preparations or with the lectins WGA, FBA or SBA. Red cells were agglutinated by all the lectins tested. Formalin-fixed bloodstream trypomastigotes bound FITC-Con A and FITC-RCA but not FITC-WAG, -SBA, -PNA, -UEA or -DBA lectins. All FITC-labeled lectins bound to red cells. Con A receptors, visualized by Con A-HRP-DAB and Con A-biotin/avidin-ferritin techniques, were distributed uniformly on T. rhodesiense bloodstream forms. No lectin receptors were visualized on control preparations. Culture procyclics lacked a cell surface coat and were agglutinated by Con A and WGA but not RCA, SBA, PP and FBP. Procyclics were not agglutinated by lectins in the presence of competing sugar at 0.25 M.(ABSTRACT TRUNCATED AT 250 WORDS)     

Generation of immune memory by haptenated derivatives of thymus-independent antigens in C57BL/6 mice. I. The differentiation of memory B lymphocytes into antibody-secreting cells depends on the nature of the thymus-independent carrier used for memory induction and/or revelation

It has been previously reported that trinitrophenylated lipopolysaccharide (TNP-LPS), a thymus-independent (TI)-1 antigen, elicits an anamnestic response to TNP in C57BL/6 mice. The ability of these mice to mount a secondary response to TI-2 antigens was analyzed. Priming with DNP-Ficoll or DNP-Dextran, both TI-2 antigens, resulted in an increased frequency of TNP-binding B lymphocytes. Evidence is presented that memory cell-induction by DNP-Ficoll does not require functional T cells. The differentiation into antibody-forming cells (AFC) of memory cells generated by DNP-Dextran or DNP-Ficoll cannot be obtained by a challenge with either antigen. There was no indication that the lack of a secondary response to TI-2 antigens was related to suppressive T cells interfering with memory expression. Memory cells induced by DNP-Dextran or DNP-Ficoll can nevertheless be activated by TNP-LPS. In contrast to the restricted sensitivity of TNP-memory cells generated by TI-2 antigens, TNP-LPS-induced memory cells are indifferently susceptible to TI-1 or TI-2 antigenic stimulation. These results are discussed in terms of memory B-cell subpopulations.     

Astroglial differentiation from neuroepithelial precursor cells of amphibian embryos: an in vivo and in vitro analysis

Initial development of astroglial phenotype has been studied in vitro in an amphibian embryo (Pleurodeles waltI), to document the differentiation potentialities acquired by neural precursor cells isolated at the early neurula stage. In particular, we sought to determine whether interactions between neuroepithelial cells and the inducing tissue, the chordamesoderm, are required beyond this stage to specify precursor cells along glial lineages. Glial cell differentiation was documented by examining the appearance of glial fibrillary acidic protein (GFAp), a specific marker of astroglial lineages. Cells expressing GFAp-immunoreactivity differentiated rapidly, after 48 hours of culture, from cultivated neural plate cells, irrespective of the presence or absence of the inducing tissue. The widespread expression of Pleurodeles GFAp protein in neural plate cultures, in which CNS precursor cells develop alone in a simple saline medium, showed that prolonged contact with chordamesodermal cells was not necessary for the emergence of the astroglial phenotype. In addition, the initial development of astroglial phenotype has been defined in vivo. The first detectable GFAp-immunoreactivity was visualized in the neural tube of stage-24 embryos, a stage corresponding to 2-3 days in culture, defining radial glial cell end-feet. Thus, dissociation and culture of neural precursor cells did not appear to modify the onset of astroglial differentiation. At stage 32, GFAp-immunoreactivity was observed over the entire length of radial glial fibers and was also evidenced in mitotic cells located in the ventricular zone, suggesting that radial glial cells were not all post-mitotic.