Chemotherapeutic efficacy of ascofuranone in Trypanosoma vivax-infected mice without glycerol

Ascofuranone, an antibiotic isolated from Ascochyta visiae, showed trypanocidal activity in Trypanosoma vivax-infected mice. A single dose of 50 mg/kg ascofuranone effectively cured the mice without the help of glycerol. Repeated administrations of this drug further enhanced its chemotherapeutic effect. After two, three, and four consecutive days treatment, the doses needed to cure the infection decreased to 25, 12, and 6 mg/kg, so that the total doses administered were 50, 36 and 24 mg/kg, respectively. Ascofuranone (50 mg/kg) also had a prophylactic effect against T. vivax infection within the first two days after administration. This prophylactic activity diminished to 80% by day 3 and completely disappeared four days after administration. Of particular interest in this study was that ascofuranone had trypanocidal activity in T. vivax-infected mice in the absence of glycerol, whereas co-administration of glycerol or repeated administrations of this drug are needed for Trypanosoma brucei brucei infection. Our present results strongly suggest that ascofuranone is also an effective tool in chemotherapy against African trypanosomiasis in domestic animals.     

The in vitro and in vivo effects of mefloquine on Trypanosoma brucei brucei.

Blood stream forms of Trypanosoma brucei brucei were grown over mouse kidney (MK) cells in minimum essential medium with various concentrations of mefloquine. The drug was observed to inhibit multiplication of the parasites in vitro. Groups of male albino mice were treated with mefloquine at 24, 48 and hours after T. b. brucei infection. Mefloquine at 0.03 mg/kg body weight administered for 4 consecutive days cleared the infection. No trypanosomes were detected in the blood of these mice for 90 days and over after the clearance of parasite from the blood. The doses for both the in vitro and in vivo therapy, were well below those prescribed for humans.     

Effects of methylglyoxal bis(ganylhydrazone) on trypanosomatid flagellates: inhibition of growth and nucleoside incorporation in Trypanosoma brucei

Methyglyoxal bis (guanylhydrazone) (MGBG) at 0.5 mM had little effect in vitro on Blastocrithidia culicis, Crithidia oncopelti, and Leishmania spp., but completely inhibited growth of Trypanosoma brucei. Inhibition became irreversible after a 3-h exposure of T. brucei culture procyclics. Treated organisms remained motile, but failed to divide. Polyamines, spermidine, and spermine, did not reverse the anti-trypanosome action of MGBG (preloading of cells or concurrent administration). Two intraperitoneal injections of the drug at a concentration of 50 mg/kg body weight at a 1-day interval greatly reduced the parasitemia of T. brucei and T. congolense in rats. Trypanosome infections, however, relapsed and killed the animals in 6 days after treatment. It was evident from the results of tracer experiments with T brucei that MGBF significantly lowered incorporation of [3H]thymidine by culture pocyclics and of [3H]uridine by bloodstream forms; in both stages [3H]leucine incorporation was only slightly inhibited. It is suggested that MGBG interferes with nucleoside incorporation by Trypanosoma and that its mode of action is different in bloodstream and culture procyclics.     

The in vitro and in vivo effects of metronidazole and chloroquine on Trypanosoma brucei brucei

Bloodstream forms of Trypanosoma brucei brucei were grown over baby hamster kidney cells in minimum essential medium with various concentrations of metronidazole (Flagyl) and chloroquine. Both drugs inhibited the multiplication of the parasite in vitro. The least effective concentrations for metronidazole and chloroquine were 0.003 mg/ml and 0.0024 mg/ml, respectively. Groups of 12-day-old female CDI mice were treated with 1 of the 2 drugs at 24, 48, and 72 hr after T. brucei infection. The drugs administered stat or daily reduced the number of parasites in the mice but did not effect a cure; they prolonged the survival period of the animals. However, metronidazole (0.1 mg/kg body weight) and chloroquine (0.08 mg/kg body weight) combined and given daily for 4 consecutive days cleared the infection. No trypanosomes were detected in the blood of these mice 3 mo after treatment. The dosages for both the in vitro (metronidazole 0.003 mg/ml; chloroquine 0.0024 mg/ml) and in vivo (metronidazole 0.1 mg/kg body weight; chloroquine 0.08 mg/kg body weight) were well below those prescribed for humans.     

Trypanosoma brucei: in vitro slender-to-stumpy differentiation of culture-adapted,monomorphic bloodstream forms

Pleomorphic Trypanosoma brucei strains are characterized by their ability to differentiate from replicating long slender forms into non-dividing short stumpy forms in the mammalian host. The differentiation process can be efficiently induced in vitro by treatment with the membrane-permeable cAMP derivative 8-(4-chlorophenylthio)-cAMP (pCPTcAMP). In contrast, monomorphic T. brucei strains do not differentiate to stumpy forms in the host. Here, we show that exposure of monomorphic, culture-adapted T. brucei bloodstream forms to pCPTcAMP allowed their subsequent differentiation into short stumpy forms. The stumpy nature of pCPTcAMP-treated parasites was confirmed by (1) morphological change, (2) inhibition of growth and DNA synthesis, (3) cell cycle arrest in the G(1)/G(0) phase, (4) expression of NADH diaphorase activity and dihydrolipoamide dehydrogenase, (5) disappearance of the small subunit of ribonucleotide reductase, (6) up-regulation of the major lysosomal membrane protein, and (7) efficient transformation into replicating procyclic insect forms after induction with citrate/cis-aconitate. Our results indicate that the inability of monomorphic T. brucei bloodstream forms to differentiate into short stumpy forms in the host may be due to a failure in the signalling pathway rather than in the differentiation process itself. Treatment of monomorphic bloodstream trypanosomes with pCPTcAMP could be a useful method for identifying the genes involved in the slender-to-stumpy differentiation process.     

Trypanosoma brucei: killing of bloodstream forms in vitro and in vivo by the cysteine proteinase inhibitor Z-phe-ala-CHN2

Cysteine proteinases were tested for their suitability as targets for chemotherapy of sleeping sickness using the peptidyl inhibitor Z-Phe-Ala-diazomethyl ketone (Z-Phe-Ala-CHN2). In vitro, the inhibitory concentration of Z-Phe-Ala-CHN;2 required to reduce the growth rate by 50% was 400 times lower for culture-adapted bloodstream forms of Trypanosoma brucei than for a mouse myeloma cell line. At an inhibitor concentration of 10;M the parasites were lysed within 48 h of incubation. Parasitemia of mice infected with T. brucei decreased to undetectable levels for 3 days following treatment with 250 mg/kg Z-Phe-Ala-CHN2 on days 3 to 6 after infection. Although parasitemia returned thereafter to control levels, infected mice treated with the inhibitor survived approximately twice as long as those treated with placebo. Z-Phe-Ala-CHN2 inhibited proteinolysis in lysosomes in vitro and almost completely blocked cysteine proteinase activity in vivo. The results demonstrate the importance of cysteine proteinase activity for survival of T. brucei and suggest that such activity is an appropriate target for antitrypanosomal chemotherapy.     

A Mitogen-activated protein kinase controls differentiation of bloodstream forms of Trypanosoma brucei

African trypanosomes undergo differentiation in order to adapt to the mammalian host and the tsetse fly vector. To characterize the role of a mitogen-activated protein (MAP) kinase homologue, TbMAPK5, in the differentiation of Trypanosoma brucei, we constructed a knockout in procyclic (insect) forms from a differentiation-competent (pleomorphic) stock. Two independent knockout clones proliferated normally in culture and were not essential for other life cycle stages in the fly. They were also able to infect immunosuppressed mice, but the peak parasitemia was 16-fold lower than that of the wild type. Differentiation of the proliferating long slender to the nonproliferating short stumpy bloodstream form is triggered by an autocrine factor, stumpy induction factor (SIF). The knockout differentiated prematurely in mice and in culture, suggestive of increased sensitivity to SIF. In contrast, a null mutant of a cell line refractory to SIF was able to proliferate normally. The differentiation phenotype was partially rescued by complementation with wild-type TbMAPK5 but exacerbated by introduction of a nonactivatable mutant form. Our results indicate a regulatory function for TbMAPK5 in the differentiation of bloodstream forms of T. brucei that might be exploitable as a target for chemotherapy against human sleeping sickness.     

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.     

Trypanosome alternative oxidase: from molecule to function

Trypanosome alternative oxidase (TAO) is the cytochrome-independent terminal oxidase of the mitochondrial electron transport chain. TAO is a diiron protein that transfers electrons from ubiquinol to oxygen, reducing the oxygen to water. The mammalian bloodstream forms of Trypanosoma brucei depend solely on TAO for respiration. The inhibition of TAO by salicylhydroxamic acid (SHAM) or ascofuranone is trypanocidal. TAO is present at a reduced level in the procyclic form of T. brucei, where it is engaged in respiration and is also needed for developmental processes. Alternative oxidases similar to TAO have been found in a wide variety of organisms but not in mammals, thus rendering TAO an important chemotherapeutic target for African trypanosomiasis.     

Development of metacyclic forms of Trypanosoma brucei sspp. in cultures containing explants of Phormia regina Meigen

When procyclic trypanosomes of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense were cultivated in Nunclon 25 cm2 flasks at 27 C in a liquid medium containing various tissue explants of Phormia regina Meigen, some of them developed into forms infective for mice. The infective stages were present at various periods of up to 29 days when the cultures were terminated. Larger numbers of explants of head-salivary glands than the other tissues used were required to produce infections. Infectivity titrations on trypanosome suspensions of T. b. brucei TRUM 252 and T. b. rhodesiense TRUM 497 indicated that only a small proportion of the populations was infective. Mice were rarely infected with trypanosomes grown in medium without explants. Only 1 mouse of the 11 inoculated developed a parasitemia from a control culture of T. b. rhodesiense TRUM 545. A few trypanosomes resembling epimastigotes and metacyclic forms were seen in stained samples of infective inocula.     

Trypanosoma brucei: infection in murine diabetes

The course of infection due to Trypanosoma brucei infection was observed in genetically diabetic and streptozotocin-induced diabetic mice. A strain of T. brucei, TREU 667, was used which produces a chronic infection in C57BL/6(B6) mice lasting greater than 60 days. Genetic diabetic mice (+db/+db) are obese, and have elevated blood glucose levels, normal levels of insulin, and impaired cell-mediated immunity. Their littermates (m+/m+, m+/+db) are of normal weight, and are normoglycemic and immunocompetent. The infected +db/+db mice lived significantly longer than the nondiabetic littermates. In contrast to this finding, streptozotocin-induced diabetic B6 mice developed higher parasitemia and had shorter survival times than control B6 mice. Continuous treatment with insulin of these streptozotocin-induced diabetic mice led to normalization of blood glucose and a significant reduction of parasitemia. While hyperglycemia may be associated with higher parasitemia and death in streptozotozin-induced diabetes, genetic factors may play an additional role in the genetic models.     

Trypanosoma brucei and T. vivax: salicylhydroxamic acid and glycerol treatment of acute and chronically infected rats

In rats infected with monomorphic Trypanosoma brucei brucei, the efficacy of the therapy with salicylhydroxamic acid plus glycerol, i.e., combined therapy, decreased with increasing time after infection. It failed completely after the infection was made chronic by suboptimal treatment for 6 weeks. When this chronic infection had been established and "optimal" treatment was given, viable trypanosomes could still be detected 1 day later in brain and muscle but not in blood. In most organs, the concentrations of salicylhydroxamic acid and glycerol were lower than in the blood plasma; the maximum concentration of glycerol in the brain was only 20% of that in plasma. The most likely explanation for the failure of the combined therapy is that, in certain tissues, the concentration of the drugs remains too low to kill extravascular trypanosomes. Other explanations, such as the selection of a resistant strain or the survival of (extravascular) forms with a more active mitochondrion, could be excluded with a high degree of probability. Suramin was very effective, even after combined therapy had failed repeatedly, while melarsoprol was less effective. As in combined therapy, the dose of melarsoprol that could cure an acute infection was insufficient to cure a chronic infection. Combined therapy failed after a spontaneous chronic infection with T. b. rhodesiense had existed for 5-7 weeks, but it was effective in T. vivax infected rats even when parasitemia had been present for at least 4 days. Effective alternative schedules for combined therapy were not found.     

Fansidar prophylaxis, therapy, and immune responses in rodent malaria (Plasmodium berghei)

In order to determine the effect of Fansidar on plasmodial infection in mice, outbred, adult, Swiss-Webster mice were treated with Fansidar (20 mg sulfadoxine and 1 mg pyrimethamine/kg body weight) at various intervals before and/or after inoculation with blood stages of Plasmodium berghei. Drug therapy resulted in cure if it was given before the parasitemia rose to 53%. Oral administration of Fansidar was more effective in reducing or preventing parasitemia than intramuscular injection. Fatal infections were prevented if mice were treated orally with one dose of Fansidar 2 days before inoculation with P. berghei, whereas only partial protection occurred in animals treated 4 or more days before inoculation. Fansidar administered on two consecutive days provided protection if the drug was given at 3 and 2 days before inoculation. Administration of Fansidar for three consecutive days protected all animals if given on days 8 to 6 before inoculation. After oral administration of Fansidar, the parasitemia dropped dramatically and was undetectable at 60 hr. At 12 hr after oral treatment, schizonts and trophozoites were numerous, but there were few merozoites. Schizonts were the predominant stage at 24 hr, whereas merozoites predominated at 36 hr. Swiss-Webster and C57BL/6 mice became immune to a lethal dose of P. berghei after 4 cycles of inoculation and drug cure. Protective immunity was still present at 472 days after the fifth parasite inoculation.     

Different types of tea products attenuate inflammation induced in Trypanosoma brucei infected mice

An in vivo study was carried out to determine the effect of different types of Kenyan tea extracts on male Swiss albino mice infected with Trypanosoma brucei brucei isolate KETRI 2710. The isolate produced a similar clinical picture after a pre-patent period of 5 days post-infection (DPI). Parasitemia levels in the untreated mice and those given different teas developed exponentially at similar rates reaching similar densities at the peak of parasitemia 8 DPI. Between 9 and 13 DPI parasitemia decreased more rapidly in tea treated compared to the untreated mice which indicated that tea lowered parasitemia level. Anaemia indicated by a fall in erythrocyte packed cell volume (PCV) occurred within 4 DPI and remained below the normal levels until the terminal stages of the disease. A significant difference (P<0.05) was observed 11 DPI between the tea treated and the untreated mice indicating that tea enhanced resistance to erythrocyte destruction. Mice treated with tea exhibited significantly (P<0.01) reduced parasite-induced hypoalbuminemia as compared to the untreated. Since albumin is a negative acute phase protein, it shows a decrease during inflammatory conditions and therefore its elevation in the mice given tea in this study clearly demonstrated that tea ameliorated inflammation induced by T. b. brucei. Although green and white teas were superior in most of these characteristics, black tea, which is the principle tea product from Kenya, displayed remarkable properties some even comparable to those of green tea. Interestingly, tea was more efficacious than dexamethasone an established anti-inflammatory drug, demonstrating its therapeutic potential.     

Morphological changes in trypanosoma brucei rhodesiense following inhibition of polyamine biosynthesis in vivo

The effect of α-difluoromethylornithine (DFMO) treatment on the morphology of African trypanosomes was investigated. For this purpose inbred mice were immunosuppressed and infected with a clone of the protozoan blood parasite Trypanosoma brucei rhodesiense. The mice were then treated with DFMO, an irreversible inhibitor of ornithine decarboxylase, which inhibits polyamine synthesis. DFMO treatment in the absence of host immunity resulted in arrest of cytokinesis of the trypanosomes and many binucleated cells could be seen in blood smears. If mice were infected with a highly virulent trypanosome clone (ETat 1.10), which does not normally transform from long slender (LS) to short stumpy (SS) forms, DFMO treatment caused SS transformation to occur on days 3–4. This morphological SS transformation was substantiated by the presence of diaphorase activity and nuclear and mitochondrial changes. The results suggest a possible involvement of polyamines in the transformation from LS to SS forms.     

Effects of Methylglyoxal bis(Guanylhydrazone) on Trypanosomatid Flagellates: Inhibition of Growth and Nucleoside Incorporation in Trypanosoma brucei*

SYNOPSIS. Methylglyoxal bis(guanylhydrazone) (MGBG) at 0.5 mm had little effect in vitro on Blastocrithidia culicis, Crithidia oncopelti, and Leishmania spp., but completely inhibited growth of Trypanosoma brucei. Inhibition became irreversible after a 3-h exposure of T. brucei culture procyclics. Treated organisms remained motile, but failed to divide. Polyamines, spermidine, and spermine, did not reverse the anti-trypanosome action of MGBG (preloading of cells or concurrent administration). Two intraperitoneal injections of the drug at a concentration of 50 mg kg body weight at a 1-day interval greatly reduced the parasitemia of T. brucei and T. congolense in rats. Trypanosome infections, however, relapsed and killed the animals in 6 days after treatment. It was evident from the results of tracer experiments with T. brucei that MGBG significantly lowered incorporation of [³H]thymidine by culture procyclics and of [³H]uridine by bloodstream forms; in both stages [³H]leucine incorporation was only slightly inhibited. It is suggested that MGBG interferes with nucleoside incorporation by Trypanosoma and that its mode of action is different in bloodstream and culture procyclics.     

Polyamine depletion following exposure to DL-alpha-difluoromethylornithine both in vivo and in vitro initiates morphological alterations and mitochondrial activation in a monomorphic strain of Trypanosoma brucei brucei

DL-alpha-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase (ODC), rapidly depletes cells of intracellular putrescine. When administered to animals and humans, DFMO cures acute infections of trypanosomiasis. In order to determine if the mechanism of drug action is related to initiation of transformation and biochemical alterations subsequent to polyamine depletion, trypanosome morphology and mitochondrial activation were studied in a monomorphic strain of Trypanosoma brucei brucei. Exposure of trypanosomes to DFMO in vivo in infected rodents or in vitro in culture resulted in a depletion of intracellular putrescine and a cessation of cell division without specific cytotoxicity. These events were followed by a transformation of the long slender bloodstream form to a short stumpy form via an intermediate morphology. Putrescine, the product of the ODC reaction, abrogates this effect. When introduced into SDM-79 medium, the intermediate form is capable of further transformation to an "insect" procyclic trypomastigote whereas the long slender form and short stumpy form are not. Short stumpy forms are incapable of binary fission and have lost their infectivity for the vertebrate host. In addition, the mitochondrial marker enzyme, NAD diaphorase, was found only in the short stumpy and intermediate forms. We hypothesize that the short stumpy phenotype may not be a viable stage in the natural transformation of the trypanosome from its mammalian host to the insect vector.     

Repeated Clorgyline Treatment Inhibits Methamphetamine- induced Behavioral Sensitization in Mice

Following the expression of the behavioral sensitization by repeated administration of methamphetamine (METH) (1 mg/kg, intraperitoneal (i.p.), once per day for five consecutive days), male ICR mice were treated with clorgyline (1 mg/kg, subcutaneous, once per day for five consecutive days), a monoamine oxidase-A inhibitor. Two hours after the final treatment with clorgyline, the mice were challenged with METH (1 mg/kg, i.p.) and locomotor activity was measured for 1 h. The mice treated with clorgyline showed a significant decrease in both vertical locomotion and horizontal rearing, compared with those treated with saline. Clorgyline treatment altered the effect of single METH challenges on apparent dopamine turnover in the cerebral cortex of the mice sensitized to METH. These results suggested a possible association of the inhibition by clorgyline of METH-induced behavioral sensitization with the alteration of dopamine turnover in the cerebral cortex of the mouse.     

Polyamine Depletion Following Exposure to DL-α-Difluoromethylornithine Both In Vivo and In Vitro Initiates Morphological Alterations and Mitochondrial Activation in a Monomorphic Strain of Trypanosoma brucei brucei

ABSTRACT. DL-α-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase (ODC), rapidly depletes cells of intracellular putrescine. When administered to animals and humans, DFMO cures acute infections of trypanosomiasis. In order to determine if the mechanism of drug action is related to initiation of transformation and biochemical alterations subsequent to polyamine depletion, trypanosome morphology and mitochondrial activation were studied in a monomorphic strain of Trypanosoma brucei brucei. Exposure of trypanosomes to DFMO in vivo in infected rodents or in vitro in culture resulted in a depletion of intracellular putrescine and a cessation of cell division without specific cytotoxicity. These events were followed by a transformation of the long slender bloodstream form to a short stumpy form via an intermediate morphology. Putrescine, the product of the ODC reaction, abrogates this effect. When introduced into SDM-79 medium, the intermediate form is capable of further transformation to an "insect" procyclic trypomastigote whereas the long slender form and short stumpy form are not. Short stumpy forms are incapable of binary fission and have lost their infectivity for the vertebrate host. In addition, the mitochondrial marker enzyme, NAD diaphorase, was found only in the short stumpy and intermediate forms. We hypothesize that the short stumpy phenotype may not be a viable stage in the natural transformation of the trypanosome from its mammalian host to the insect vector.