Trypanosoma brucei: biochemical and morphological studies of cytotoxicity caused by normal human serum

The biochemical and morphological events which accompany lysis of Trypanosoma brucei by normal human serum have been described. The prelytic events include loss of infectivity and rapid cation shifts across the cell membrane. This is followed by cell swelling, fraying of the surface coat of the cell, loss of intracellular organelles, and eventually cell lysis. The data presented are consistent with a colloid osmotic mechanism of lysis induced by irreversible acute damage to the normal permeability properties of the trypanosome plasma membrane.     

Trypanosoma cruzi: in vitro interactions between cultured amastigotes and human skin-muscle cells.

Established cultures of human skin-muscle cells were used for determining the parasite—host cell relationship of Trypanosoma cruzi amastigotes (12–16 passages) cultured in a cell-free medium (F-69) at 37 C. The medium used for this experiment was tissue culture fluid M-199 enriched with 10% fetal bovine serum and relatively high concentrations of ATP, ADP and AMP. Amastigotes entered skin-muscle cells incubated at 32 or 35 C, multiplied and completed their intracellular life cycle in about 7 days. At 35 C, 23.6% of cells became infected in 7 days and at 32 C, 43.6% were infected in 5 days. The higher infection rate of cultured cells at 32 C was probably due to more frequent and prolonged cell-parasite contact, as amastigotes multiplied in the tissue culture medium and remained viable for a longer period at the lower temperature. As a control, epimastigotes were used to infect skinmuscle cells. Epimastigotes transformed into metacyclic trypomastigotes before entering host cells, multiplied, and completed the intracellular life cycle. We conclude that the amastigotes cultured in F-69 at 37 C are biologically similar to intracellular amastigotes from the vertebrate host, in that both can multiply and complete the life cycle intracellulary.     

Trypanosoma brucei: New radioisotope assay for quantitating cell lysis

A new radioisotope technique for quantitatively determining the extent of cell lysis of trypanosomes is described. Trypanosoma brucei was pulse-labeled with [³H]leucine and then incubated at 37 C in lytic (human serum) or nonlytic (rabbit serum) media. The release into the incubation medium of radioactively labeled intracellular macromolecules was then determined. A linear relationship was found between the release of TCA-precipitable radioactivity and lysis, as determined by conventional hemacytometer counting. This method is used to quantitate the trypanocidal activity in normal human serum.     

Trypanosoma congolense: isolation and purification.

Yields of Trypanosoma congolense grown in rats may be increased by placing the rats in a 37 °C environment for 1 hr prior to sacrifice. A further increase in the number of parasites recovered per rat may be achieved by replacement of blood removed by a lactated Ringer's solution with 5% glucose as the rat is being bled from the abdominal aorta. The Ringer's solution serves to maintain intravascular volume during the bleeding procedure and thereby prevents premature cardiac arrest. Erythrocytes in infected blood may be then lysed by raising and rapidly lowering the osmolarity of the blood. This permits separation of the trypanosomes from 95% of the erythrocytes by differential centrifugation. The remaining blood cell contamination may then be removed on a small DEAE-cellulose column. The purified trypanosomes are motile, infective, and intact as judged by electron microscopy. More than 10¹⁰ purified T. congolense can be obtained from three adult rats by these methods.     

Trypanosoma cruzi: cultivation in macromolecule-free semisynthetic and synthetic media.

A macromolecule-free semi-synthetic medium (F-81) was devised to culture Trypanosoma cruzi serially at room temperature. F-81 contains only one undefined substance, trypticase, which consists primarily of short-chain polypeptides. In F-81 medium T. cruzi will grow to a density of 35 to 43 × 10⁶ organisms/ml, a density comparable to that obtainable in a serum-containing medium such as F-69. High concentrations of water-soluble vitamins appear to have a serum-replacing effect in the F-81 medium. A completely synthetic medium (F-84) was prepared by replacing trypticase in F-81 with Trager's amino acid mixture. T. cruzi epimastigotes could be serially cultured in F-84, with a maximum yield of 9.2 × 10⁶ organisms/ml of medium after 3 to 4 weeks of incubation at 27 C.     

Trypanosoma brucei: trypanocidal effect of salicylhydroxamic acid plus glycerol in infected rats.

Rats inoculated with Trypanosoma brucei brucei EATRO 427 and having a high degree of parasitemia were treated with a series of intra-peritoneal injections of Salicylhydroxamic acid (SHAM) plus glycerol. Permanent cures were obtained with 380 mg/kg SHAM plus 3.8 g/kg glycerol, a dosage regime which was just sublethal. Using a regime with which permanent cure was obtained, the SHAM concentration in the blood plasma remained above 2 mmole/liter for about 20 min, while the glycerol concentration remained above 22 mmole/liter for about 1 hr. The brain concentration of SHAM was close to the plasma concentration. The concentration of glycerol in the brain remained far below the plasma concentration, reaching 6 to 8 mmole/liter between 1 and 2 hr after the beginning of treatment. Treatment with glycerol did not affect the mobility of the trypanosomes nor the survival of infected rats after treatment with suramin.     

Trypanosoma congolense: Surface glycoproteins of two early bloodstream variants: III. Immunochemical characterization

A radioimmunoassay (RIA) for the variant-specific glycoproteins (VSG-1 and VSG-2) of two sequentially appearing variants of Trypanosoma congolense has been devised. When the isoelectrically focused VSG-1 components (VSG-1a, VSG-1b, and VSG-1c) are used as inhibitors of the VSG-1-anti-VSG-1 interaction, the RIA inhibition curves resemble each other, although minor differences in the high-affinity region of the curves can be detected. The heterologous antigen (VSG-2) does not inhibit the VSG-1-anti-VSG-1 interaction except at very high concentrations, indicating there is little cross-reactivity between highly purified VSG-1 and VSG-2. Nevertheless, heterologous antiserum, directed against VSG-2, will inhibit the VSG-1 -anti-VSG-1 interaction, and this property is shared to a significant degree by rabbit antiserum directed against an unrelated antigen. We have interpreted these findings as suggesting that: (1) there may be a constant region common to both VSG proteins, and (2) the constant region of the immunoglobulin molecule may also bind VSG proteins. Preliminary experiments show that the VSG-1 molecule augments binding of the Clq component of complement to the Fc region of immunoglobulin G.     

Trypanosoma brucei: host parasite interaction in parasite destruction by salicylhydroxamic acid and glycerol in mice

Mouse blood and serum contain a synergistic factor which affects both the speed and completeness of destruction of Trypanosoma brucei in the presence of salicylhydroxamic acid (SHAM) and glycerol. The action of this factor is dose dependent producing complete killing in infected whole blood with 2 mM SHAM and 12 mM glycerol but not in a mixture of 20% infected whole blood and 80% buffer containing the same final concentration of SHAM and glycerol. This factor may account for the discrepancy in reports showing that SHAM-glycerol does not kill 100% of the exposed parasites in vitro yet is able to cure infected animals. The factor is not due to an acquired immune response, complement action, nor lipoproteins. Should the level of the factor be able to be increased, this could greatly increase the effectiveness of SHAM-glycerol.     

Trypanosoma cruzi: identification of a cell surface polysaccharide.

An immunogenic polysaccharide prepared by phenol-water extraction of Trypanosoma cruzi was characterized by polyacrylamide gel electrophoresis and molecular sieve chromatography. The polysaccharide was shown to be a cell surface constituent by adsorption of rabbit anti-polysaccharide serum with live culture forms of the protozoa. The cell surface localization of the antigen was visualized using fluorescein- and ferritin-conjugated antibodies.     

Trypanosoma cruzi: defined medium for continuous cultivation of virulent parasites.

A liquid medium was developed for the continuous cultivation of Trypanosoma cruzi. Among the several highly purified macromolecules tested only bovine liver catalase, horseradish peroxidase, lactoperoxidase, and bovine hemoglobin supported the continuous growth, at high yield, of mice-virulent Trypanosoma cruzi; other hemoproteins were inactive. Bovine liver catalase showed optimal Trypanosoma cruzi growth-promoting activity, parasites reaching 20 × 10⁶ parasites/ml (95% epimastigotes) at about 10 days in most of the 45 subpassages to date. Furthermore, this protein in the incubation medium provided all the amino acid requirements of actively growing parasites, thus eliminating the need for exogeneous free amino acids. Additional experiments revealed that the hemoprotein's growth-promoting activity was independent of any enzymatic activity and that reconstituting the exact protein composition by means of exogeneous amino acids did not support parasite multiplication, suggesting the importance of the primary structure of the active proteins for growth-promoting activity. These active macromolecules supported the multiplication of five different strains of Trypanosoma cruzi, but did not support Leishmania brasiliensis or Leishmania mexicana proliferation, suggesting species specificity.     

Trypanosoma cruzi: intracellular stages grown in a cell-free medium at 37 C.

A liquid medium containing a high concentration of water-soluble vitamins and ATP was developed for serial cultivation of Trypanosoma cruzi at 27–37 C; fetal bovine serum and trypticase were the only undefined substances in this medium. At 27 C, Trypanosoma cruzi grows primarily (over 99%) as epimastigotes with a population density reaching 92.7 × 10⁶/ml after 12 days of incubation. During the first subculture at 37 C, many epimastigotes from the original inocula changed into metacyclic trypomastigotes after 48 hr; the trypomastigotes subsequently transformed into amastigotes by 96 hr. In the second passage at 48 hr, 57.8% of the organisms were trypomastigotes which changed into amastigotes by the end of the incubation period. The proportion of amastigotes in the third and subsequent passages increased steadily as the proportion of epimastigotes gradually diminished. Amastigotes thus obtained could be serially subcultured indefinitely, yielding population densities of over 3.0 × 10⁷/ml of medium in 4–5 days at 37 C. Available evidence indicates that these amastigotes are morphologically and physiologically similar to intracellular amastigotes.     

Trypanosoma brucei: recognition in vitro of two developmental forms by murine macrophages

In vitro, murine macrophages attach to the midgut form of Trypanosoma brucei in the absence of exogenous proteins. Recognition appears to be specific and saturable. Attachment is mediated by a non-Fc, non-C3 receptor on the macrophage plasma membrane. The attachment mechanism is protease sensitive, temperature sensitive, requires the presence of divalent cations, and is functional in fetal calf serum-free medium. The midgut form is also lysed in normal rabbit serum by activating the alternative complement pathway. The form isolated from the blood is neither lysed in normal serum nor is it spontaneously recognized by the macrophage, in vitro. Partial trypsinization of the bloodstream form, however, results in both the triggering of alternative complement pathway lysis and spontaneous uptake into macrophages. Murine macrophages attach to the midgut form of T. brucei by a receptor on the macrophage plasma membrane which is capable of recognizing particulate activators of the alternative complement pathway.     

Trypanosoma rhodesiense: semiautomated microtesting for quantitation of antitrypanosomal activity in vitro

A rapid, semiautomated microdilution method was developed to measure the antitrypanosomal activity of large numbers of compounds against blood form Trypanosoma rhodesiense at 37 C in vitro. Parasites harvested from infected rats were incubated for 3 hr in microtitration plates with serial dilutions of test compounds. Inhibition of uptake of radiolabeled thymidine and l-leucine by the parasites served as indicators of antitrypanosomal activity. Repeated measurements of the activity of ethidium bromide demonstrated the sensitivity and precision of the method. Several additional antitrypanosomal drugs were tested in vitro and all except tryparsamide (a pentavalent arsenical) and suramin (a complex polyanion) were consistent with in vivo activity. Antimicrobial agents which are not effective antitrypanosomal drugs failed to show activity in vitro. Other compounds active in vitro included allopurinol and a number of aromatic diamidines, carbamodithioic acid derivatives, and 2-acetylpyridine thiosemicarbazones. This in vitro microtest system was developed as a primary screen for the selection of active compounds in a new Antitrypanosomal Drug Development Program.     

Trypanosoma cruzi: isolation and characterization of membrane and flagellar fractions.

A cell fractionation procedure for obtaining membrane and flagellar fractions was developed using Trypanosoma cruzi epimastigote forms. The cells, swollen in an hypotonic medium, were disrupted in the presence of a nonionic detergent, and fractions were isolated by differential centrifugation. The flagellar fraction, pelleted in 10 min at 10,000g, was further purified on a sucrose gradient. The membrane fraction was obtained by centrifugation of the supernatant at 27,000g for 30 min. Electron microscopy of the isolated fractions demonstrated a high degree of purity of each fraction. The membrane fraction showed homogeneous vesicles with low ribosome content. In frozen-etched preparations, the distribution of intramembranous particles on the vesicles was similar to that of the plasma membrane of intact cells. Enzymatic assays indicated that the membrane and flagellar fractions had low contamination with mitochondria and lysosomes. 5′-Nucleotidase activity was not detected in the membrane fraction; Mg²⁺-dependent ATPase activity was slightly enhanced, although, the enzyme was not sensitive to Na⁺, K⁺, and Ca²⁺ ions. The membrane fraction showed about five times the adenylyl cyclase activity of the whole homogenate. Gel immunodiffusion revealed the whole antigen of T. cruzi extracted by formamide to be identical to the membrane fraction when both were tested against rabbit anti- T. cruzi (epimastigote) immune serum.     

Trypanosoma cruzi: ultrastructural cytochemistry of mitochondrial enzymes

Mitochondrial enzymes were detected cytochemically in all developmental stages of Trypanosoma cruzi maintained in tissue cultures at the light and electron microscope levels. Cytochrome oxidase (CO) was detected using the diaminobenzidine method. Succinate dehydrogenase (SDH), isocitrate dehydrogenase (ICDH), NADPH diaphorase, α-glycerophosphate dehydrogenase (GPDH), and β-hydroxybutyrate dehydrogenase (HBDH) were detected using the dystyril nitroblue tetrazolium salt. A reaction product indicative of CO, SDH, ICDH, and NADPH diaphorase activities was found either in the inner mitochondrial membrane or in the cristae. β-HBDH and α-GPDH activities, however, were localized only in the inner membrane. No difference in the localization and intensity of the reaction was observed in the various stages of T. cruzi.     

Trypanosoma brucei: morphometric changes and loss of infectivity during transformation of bloodstream forms to procyclic culture forms in vitro.

The transformation of Trypanosoma brucei bloodstream forms to procyclic culture forms in the semidefined medium SDM-77 has been studied by light microscopy and quantitative electron microscopy. Stumpy and intermediate forms are able to transform to culture forms whereas slender forms die after approximately 24 hr. The surface coat and infectivity for the mammalian host are lost after 72 hr. Morphometrical analysis of the cells during transformation process revealed: (1) The cytoplasm and the cell surface increased significantly; (2) the volume density of the mitochondrion increased twofold and the surface density of the inner mitochondrial membrane increased threefold; (3) the volume density of the glycosomes remained about constant; (4) the volume density of the lipid inclusions increased up to 72 hr, probably as a result of the complete oxidation of glucose. Transformation as observed by light microscopy was completed in 72 hr. However, quantitative electron microscopy revealed that establishment of the culture form was incomplete even after 11 days.     

Trypanosoma cruzi: allopurinol in the treatment of mice with experimental acute Chagas disease

The therapeutic effect of allopurinol was studied in an experimental Trypanosoma cruzi infection (Chagas disease) in outbred IVIC-NMRI and inbred C57B1/6J mice intraperitoneally inoculated with the parasites 2–6 days before drug treatment. Allopurinol protected against T. cruzi infection. This effect was evidenced by highly significant reductions in both parasitemias and mortality rates and increased survival time in allopurinol-treated animals compared with untreated infected mice. Allopurinol protected effectively when administered in 10 daily doses of 32–64 mg/kg body wt/day injected intraperitoneally. Using direct methods, parasitemia remained undetectable for at least 310 days. An indirect method, subinoculation to susceptible mice, showed a few circulating trypanosomes which decreased greatly in number after a second schedule of allopurinol treatment; finally no trypanosomes were detectable 275 days after treatment initiation. Allopurinol also induced a strong trypanostatic effect when tested in vitro on five different Trypanosoma cruzi strains (optimal inhibitory concentration: 3 μg/ml). These results suggest that allopurinol protects mice with acute Chagas infection by a direct trypanostatic effect. The low toxicity of this drug suggests its use in more chronic experimental Chagas infections.     

Trypanosoma cruzi: reversal of inhibition of host muscle differentiation after exposure to elevated temperatures

The L6E9 myoblast cell line can be grown as individual cells in "growth medium," or can be induced to fuse and differentiate to form multinucleated myotubes either at 37 C or at 40.5 C in "differentiation medium." It has previously been shown that myoblasts with infected Trypanosoma cruzi (Brazil strain) cannot differentiate to form myotubes. Moreover, the mRNAs for contractile proteins are not induced in these infected cells. Infected myoblasts grown in "differentiation medium" at 37C were unable to differentiate by 7 days. The infection was maintained at 100%, and the number of trypomastigotes in the supernatant increased with time (peak greater than 10(6)/ml). At 40.5C, however, infected myoblasts gradually eliminated their infection. The percentage of parasitized cells was reduced to less than 1% by the 7th day of observation. There was also a decrease in the number of trypomastigotes in the supernatant. Moreover, significant fusion was observed in these cultures by morphological criteria. Using 32P-labeled recombinant DNA probes, it was shown that, at 37C, there was an inhibition of mRNAs for muscle-specific contractile proteins (myosin heavy chain and alpha-actin), whereas nonspecific mRNAs were not inhibited. Furthermore, infected myoblasts exposed to 40.5C exhibited no inhibition of mRNAs for myosin heavy chain and alpha-actin. Myoblasts cleared of their infection could readily be reinfected. This study demonstrates that the inhibition of muscle differentiation induced by T. cruzi is reversible when cultures are exposed to elevated temperatures.     

Trypanosoma congolense: calf erythrocyte survival.

Hereford calves infected with Trypanosoma congolense developed an anemia which was most severe 10 weeks after infection when packed cell volumes (PCV) averaged 21.1 ± 2.5% (±2 SE) as compared to 33.1 ± 2.1% for controls. At the termination of the study, at 28 weeks postinfection PCVs of infected animals had risen to 27.5 ± 1.0% as compared to 34.0 ± 1.7% for controls. In parallel with PCVs the apparent half-lives of ⁵¹Cr-labeled erythrocytes were reduced as early as the first 2 weeks postinfection. The greatest difference in erythrocyte half-lives between infected and control animals was found during the fourth to sixth weeks of infection when volumes for infected animals averaged 128 ± 46 hr as compared to 321 ± 30 hr for controls. During this period the parasitemia was at its highest level. At 28 weeks postinfection the average apparent half-life of infected animals was 243 ± 43 hr compared with 304 ± 11 hr for controls. No differences were observed in gastrointestinal loss of ⁵¹Cr between infected and control animals; however, urinary excretion of isotope was greatly increased in infected animals when compared to controls. No significant changes in total blood volumes were observed between infected and control animals but total plasma volumes increased and total erythrocyte volumes decreased significantly in infected animals.     

Trypanosoma congolense: surface glycoproteins of two early bloodstream variants. I. Production of a relapsing infection in rodents

A strain of Trypanosoma congolense has been cloned, passaged through the tsetse fly, and subsequently recloned. Relapsing infections have been induced in two rats by syringe passage of the cloned trypanosomes. The variant-specific glycoprotein of the initial cloned variant (VSG-1) and those from the two different variants produced in the two relapsing infections (VSG-2 and VSG-3) may be distinguished from each other by their isoelectric-focusing patterns. In this experimental system, cloned T. congolense, like Trypanosoma brucei, undergoes antigenic variation; the conversion of the VSG-1 into the VSG-2 isoelectric-focusing spectrotype was followed. These VSGs may be the products of sequentially expressed genes.