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 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.     

Effect of Trypanosoma brucei brucei on erythropoiesis in infected rats

Anemia generated from African trypanosome infection is considered an important symptom in humans and in domestic animals. In order to recover from anemia, the process of erythropoiesis is essential. Erythropoiesis is affected by erythropoietin (EPO), an erythropoietic hormone, supplying iron and inflammatory and proinflammatory cytokines. However, the role of these factors in erythropoiesis during African trypanosome infection remains unclear. In the present study, we analyze how erythropoiesis is altered in anemic Trypanosoma brucei brucei (interleukin-tat 1.4 strain [ILS])-infected rats. We report that the packed cell volume (PCV) of blood from ILS-infected rats was significantly lower 4 days after infection, whereas the number of reticulocytes, as an index of erythropoiesis, did not increase. The level of EPO mRNA in ILS-infected rats did not increase from the third day to the sixth day after infection, the same time that the PCV decreased. Kidney cells of uninfected rats cultured with ILS trypanosome strain for 8 hr in vitro decreased EPO mRNA levels. Treatment of both ILS and cobalt chloride mimicked hypoxia, which restrained the EPO-production-promoting effect of the cobalt. Messenger RNA levels of β-globin and transferrin receptor, as markers of erythropoiesis in the bone marrow, also decreased in ILS-infected rats. Levels of hepcidin mRNA, which controls the supply of iron to the marrow in liver, were increased in ILS-infected rats; however, the concentration of serum iron did not change. Furthermore, mRNA levels of interleukin-12, interferon-γ, tumor necrosis factor-α, and macrophage migration inhibitory factor in the spleen, factors that have the potential to restrain erythropoiesis in bone marrow, were elevated in the ILS-infected rats. These results suggest that ILS infection in rats affect erythropoiesis, which responds by decreasing EPO production and restraining its function in the bone marrow.     

Tissue alterations in Microtus montanus chronically infected with Trypanosoma brucei gambiense

Changes in liver, spleen, kidneys, heart, and brain are reported for Microtus montanus chronically infected with Trypanosoma brucei gambiense. An increase in body weight of infected animals was attributable to a significant increase in total mass of spleen, liver and kidney. Cellular infiltrate consisting primarily of lymphocytes and plasma cells was observed in all organs and was particularly evident in intralobular connective tissue of the liver, adipose tissue of the hilum, and adjacent medullary region of the kidney, spleen, and the meninges. Disruption of normal metabolism and the pathological changes observed in liver and kidney suggest that the survival of trypanosome-infected voles is dependent largely on the physiological response occurring in these organs.     

Elevated serum and hepatic tyrosine aminotransferase in voles chronically infected with Trypanosoma brucei gambiense.

Chronic infections of voles (Microtus montanus) with Trypanosoma brucei gambiense result in marked elevations in both serum and hepatic tyrosine aminotransferase. Serum enzyme levels correlate poorly with hepatic levels but well with parasitemia. Because the trypanosome itself contains high levels of this same enzyme, it is hypothesized that lysis of extravascular parasites, possibly due to agglutination by variant-specific antibody, releases parasite enzymes into host tissues, resulting in the observed increase in serum enzyme levels. The possible role of increased tyrosine transamination in the behavioral syndrome of trypanosome-infected voles is considered.     

Multiple alpha-keto aciduria in Microtus montanus chronically infected with Trypanosoma brucei gambiense

Microtus montanus chronically infected with a monomorphic strain of Trypanosoma brucei gambiense excreted in urine greatly elevated quantities of not only the aromatic alpha-keto acids, phenylpyruvic and 4-hydroxyphenylpyruvic acids, but also two aliphatic alpha-keto acids, pyruvic and alpha-ketoglutaric acids. Elevated keto acid excretion began approximately midway through infection and quantities remained elevated until death. Daily keto acid excretion did not correlate with daily parasitemia. Thus, a large metabolic disturbance exists in laboratory animals infected with African trypanosomes. The multiple alpha-keto aciduria potentially contributes to the pathogenesis of chronic African trypanosomiasis.     

Increased urinary excretion of aromatic amino acid catabolites by Microtus montanus chronically infected with Trypanosoma brucei gambiense

Microtus montanus infected with Trypanosoma brucei gambiense for 16 and 21 days excreted significantly greater quantities of several aromatic amino acid catabolites when compared to uninfected control animals. Very large quantities of three aromatic alpha-keto acids (alpha-oxocarboxylic acids), phenylpyruvic acid, 4- hydroxyphenylpyruvic acid and indole-3-pyruvic acid, were excreted by infected animals. Increased excretion of indole-3-lactic acid and indole-3-acetic acid was also detected. Gas chromatographic-mass spectral analysis of the trimethylsilyl derivatives of phenylpyruvic acid, 4- hydroxyphenylpyruvic acid and indole-3-pyruvic acid confirms the identity of the aromatic alpha-keto acids elevated during infection. The marked alpha-keto aciduria indicates that a large disturbance exists in aromatic amino acid metabolism in this chronic animal model of African trypanosomiasis. The disturbance may contribute to the pathogenesis of the disease. The increased catabolite concentrations may also prove to be useful diagnostically and prognostically.     

The phosphoglucose isomerases of the bloodstream forms of Trypanosoma brucei and Trypanosoma vivax.

1. The phosphoglucose isomerases (PGI's) of the bloodstream forms of Trypanosoma brucei and T. vivax have been purified some 150-fold, using cellulose ion-exchange chromatography, gel filtration and isoelectric focussing. 2. The two trypanosome enzymes showed many similarities in kinetic properties, but differed from each other somewhat in thermal stability and in isoelectric point. 3. Both trypanosome enzymes differ from PGI's from other sources in having a higher Ki for the competitive inhibitor 6-phosphogluconate.     

Inducible nitric oxide synthase and nitrotyrosine in the central nervous system of mice chronically infected with Trypanosoma brucei brucei

Human African trypanosomiasis, or sleeping sickness, evolves toward a meningoencephalitic stage, with a breakage in the blood-brain barrier, perivascular infiltrates, and astrocytosis. The involvement of nitric oxide (NO) has been evoked in the pathogenic development of the illness, since NO was found to be increased in the brain of animals infected with Trypanosoma brucei (T. b.) brucei. An excessive NO production can lead to alterations of neuronal signaling and to cell damage through the cytotoxicity of NO and its derivatives, especially peroxynitrites. In African trypanosomiasis, the sites of NO production and its role in the pathogenicity of lesions in the central nervous system (CNS) are unknown. In a chronic model of African trypanosomiasis (mice infected with T. b. brucei surviving with episodic suramin administration), NADPH-diaphorase staining of brain slides revealed that NO synthase (NOS) activity is located not only in endothelial cells, choroid plexus ependymal cells, and neurons as in control mice but also in mononuclear inflammatory cells located in perivascular and parenchyma infiltrates. An immunohistochemical study showed that the mononuclear inflammatory cells expressed an inducible NOS activity. Furthermore, the presence of nitrotyrosine in inflammatory lesions demonstrated an increased NO production and the intermediate formation of peroxynitrites. The detection of extensive formation of nitrotyrosine in the CNS parenchyma was observed in mice having shown neurological disorders, suggesting the role of peroxynitrites in the appearance of neurological troubles. In conclusion, this study confirmed the increased NO synthesis in the CNS of mice infected with T. b. brucei and suggests a deleterious role for NO, through the formation of peroxynitrites, in the pathogenesis of African CNS trypanosomiasis.     

Neurochemical and activity changes in rats infected with Trypanosoma brucei gambiense

The brains of rats infected with Trypanosoma brucei gambiense were analyzed for their content of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), serotonin, and 5-hydroxyindoleacetic acid (5-HIAA). In the caudate of infected rats, dopamine levels increased by 34% and 5-HIAA levels by 20%. DOPAC levels declined by 30% in the caudate, but this change was significant at only P less than 0.1. In the pons/medulla of infected rats the serotonin concentration dropped by 21% and the 5-HIAA concentration rose by 25%. Activity levels in infected rats decreased by 49% (gross movement) and 44% (total movement) relative to those of uninfected control rats.     

Cytokines and the acute phase response in post-treatment reactive encephalopathy of Trypanosoma brucei brucei infected mice

Stimulation of the acute phase response during infection of mice with Trypanosoma brucei brucei (T. b. brucei) was investigated in an experimental model of the post-treatment reactive encephalopathy (PTRE), a common side-effect of anti-trypanosome therapy. Plasma levels of the acute phase proteins (APP), haptoglobin (Hp) and serum amyloid P (SAP) increased by day 7 post-infection, but by day 20 had fallen to an intermediate level. This was accompanied by induction of the cytokines, interleukin (IL)-6 and tumour necrosis factor-alpha (TNFalpha) in both liver and brain. Treatment of mice on day 21 with a subcurative dose of diminazene aceturate (Berenil), a procedure known to induce a mild PTRE, cleared the parasite from the circulation with plasma APP and liver expression of mRNA for IL-6 and TNFalpha returning to the levels in the controls. Cytokine mRNA for both IL-6 and TNFalpha was detected in the brains of animals with developing PTRE although TNFalpha was not significantly greater than in the control group. A further subcurative dose of Berenil, leading to a more severe PTRE, was associated with elevated serum concentrations of Hp and SAP, increased TNFalpha mRNA in the liver and detectable IL-6 and TNFalpha mRNA in the brain. mRNA for IL-1alpha was expressed in brain and liver samples from all animals. A severe PTRE caused a systemic acute phase response which was not apparent with a mild PTRE. The pattern of cytokine mRNA induction was similar following both drug treatments. However, the difference in APP production could be caused by a breakdown in the blood-brain barrier during severe PTRE allowing cytokine synthesised in the brain to enter the circulation and maintain a systemic response.     

Trypanosoma brucei brucei, T. brucei gambiense, and T. brucei rhodesiense: common glycoproteins and glycoprotein oligosaccharide heterogeneity identified by lectin affinity blotting and endoglycosidase H treatment

Whole cell extracts of 10 clones of bloodstream forms of African trypanosomes representing two strains of Trypanosoma brucei gambiense, one strain of T. b. rhodesiense and one strain of T. b. brucei were fractionated on sodium dodecyl sulfate-polyacrylamide gels, electrophoretically transferred to nitrocellulose paper, and probed with horseradish peroxidase conjugated lectins to detect glycoproteins. Variant specific glycoproteins of all 10 clones bound peroxidase labeled concanavalin A, but peroxidase labeled wheat germ agglutinin bound to the variant specific glycoproteins of only 3 of the 10 clones examined. In addition, 22 other glycoproteins expressed in common by all clones bound peroxidase labeled concanavalin A; 19 common glycoproteins bound peroxidase labeled wheat germ agglutinin. Lectin binding to transferred glycoproteins was specifically inhibited by appropriate monosaccharides, alpha-methyl mannoside for concanavalin A and N-acetyl glucosamine for wheat germ agglutinin. Prior incubation of blots in endo-beta-N-acetylglucosaminidase H eliminated binding of peroxidase-labeled concanavalin A to most of the 22 common glycoproteins. Two glycoproteins, designated Gp 81 and Gp 110, were the major Endoglycosidase H resistant components. Endoglycosidase H treatment also reduced binding of peroxidase labeled concanavalin A to the variant specific glycoproteins of 7 clones. The variant specific glycoproteins from the 3 clones that bound peroxidase labeled concanavalin A following enzyme treatment were those that bound peroxidase labeled wheat germ agglutinin. These results show that African trypanosomes express a greater number of glycoproteins than has been reported previously and that only a limited number of these glycoproteins bear Endoglycosidase H resistant oligosaccharides.     

Suppressor cells in mice infected with Trypanosoma brucei.

Within 2 to 3 days of infection with Trypanosoma brucei strain S42, the ability of spleen cells from infected CBA mice to mount a primary in vitro antibody response to sheep red blood cells (SRBC) is profoundly reduced, and suppressor cells are generated as detected by cell mixture experiments. Suppressor cell activity lies in the T and adherent cell compartments of spleens from infected mice, but not in the B cell compartment, although antibody responses to a thymus-independent antigen, DNP-Ficoll, are significantly reduced. Suppression of antibody responses of normal spleen cells depends on viable cells from infected mice. The trypanosome, itself, plays no direct role in suppression, and we have ruled out the possibility of antigenic competition as a mechanism of suppression. Our data is consistent with the model of suppressor T cells induced by concanavalin A mitogenesis. We hypothesize that trypanosome antigens may directly stimulate T cells with the concomitant release of factors with affinity for macrophage surfaces thus becoming suppressive for T and B cell responses.     

Evidence for glycerol 3-phosphate:glucose transphosphorylase activity in bloodstream Trypanosoma brucei brucei

1. Anaerobic glycolysis in intact bloodstream Trypanosoma brucei brucei was studied. 2. Fructose, glucose and mammose were aerobically catabolized at rates of 3.4, 3.0 and 2.5 and anaerobically at rates of 0.38, 2.75 and 2.35 mumol hexose/hr/10(8) trypanosomes respectively. 3. Glycerol 3-phosphate and ADP accumulated approximately to the same level from anaerobic catabolism of the three hexoses. However, fructose catabolism stopped within 15-20 min but addition of glucose to these already immobilized trypanosomes temporarily caused a rapid characteristic drop in glycerol 3-phosphate level at a rate of 40 nmol/min/10(8) trypanosomes and correspondingly glucose 6-phosphate, glycerol and pyruvate levels were raised. 4. These observations are not consistent with the proposed requirements for the reverse glycerol kinase in anaerobic net ATP production. Instead, we propose a glycerol 3-phosphate:glucose transphosphorylase that catalyses the formation of glycerol and glucose 6-phosphate.     

Changes in albumin levels in blood and urine of Microtus montanus chronically infected with Trypanosoma brucei gambiense

Serum albumin and glucose concentrations and urinary excretion of alpha-keto acids and proteins were determined in samples obtained throughout a chronic Trypanosoma brucei gambiense infection in Microtus montanus. An increase in urinary excretion of alpha-keto acids and proteins during the terminal stage of disease was accompanied by a decrease in serum glucose concentration. This terminal hypoglycemia reflected a depletion of liver glycogen in most animals. In contrast (and the major focus of this study) serum albumin concentration was decreased by the second week of infection and in the final sample obtained was less than 50% of that measured in preinfection samples. Female animals survived approximately 1 wk longer than males and were less susceptible during the acute phase of disease. This relative resistance was most likely due to the fact that female animals were relatively more efficient in limiting parasitemia during the first week of infection. The similarity between humans and voles in terms of protein and alpha-keto acid excretion and changes in serum concentrations of glucose and albumin during trypanosome infection further validate the use of Microtus as an experimental model for trypanosomiasis in humans.     

Disruption of spermatogenesis in boars sub-clinically infected with Trypanosoma brucei brucei

Data from 14 crossbred (Landreace x Large white) boars aged 10-12 months were used to investigate specific germ cells and to what extent Sertoli cells are prone to sub-clinical infection with strain Y58/98 Trypanosome brucei brucei and effects on spermatogenesis. Boars were divided into three groups, A, B and C of 5, 5 and 4 animals, respectively. Groups A and B were infected intraperitoneally with 2.8x10(6) trypanosomes per animal. Group C consisted of intact controls. At stable sub-clinical trypanosomiasis, boars in groups A and B together with two from the controls were weighed, scrotal circumferences were measured and animals were castrated on days 56 and 84 post infection, respectively. Testes were weighed. A portion of a testis was processed for histomorphometric assessment and another portion was used to determine gonadal sperm reserves by haemocytometry. Crude cells were converted to true cells.Sub-clinical trypanosomiasis was characterised by low live and testes weights, reduced scrotal circumference, scanty parasitemia peaks at long intervals and decreased libido. Histomorphometry of animals infected with T. brucei brucei revealed somniferous tubular distortion, denudation and or degeneration of germ cells and Sertoli cells leading to distortion of spermatogenesis. Spermatids and young primary spermatocytes were most prone to, while Sertoli cells and spermatogonia were least affected by sub-clinical trypanosomiasis. There was evidence of regeneration of germ cells from precursor stem cells, resulting in slightly increased gonodal sperm reserves as the post infection period increased. Infected boars may not attain original fertility levels consequently. It was concluded that boars in tropical regions that harbour endemic disease should be maintained under prophylactic conditions.     

Detection and quantification of variant specific antigen in the plasma of rats and mice infected with Trypanosoma brucei brucei

Variant specific antigen (VSA), the principal constituent of the surface coat of salivarian trypanosomes, was detected by gel immunoassays in the plasma of rats and mice infected with Trypanosoma brucei brucei. The quantity of VSA in plasma was measured in radial immunodiffusion tests using a monospecific antiserum and purified VSA as a standard. During the first peak of parasitemia, a statistically significant, linear relationship was determined between the number of parasites in the blood (in the range between 4 x 10(8) and 10(9)/ml) and the concentration of VSA in the plasma (28-320 microgram/ml). The VSA from parasites of the first peak was lost within 2 days of remission. Variant antigens of parasites constituting the second peak then began to appear in the plasma of infected rats. All plasma samples had been separated from parasites and blood cells within 15 min of blood collection. The pH of plasma was controlled with a buffered anticoagulant. No soluble parasite antigens, other than VSA, were detected in the plasma of infected hosts. The results of this study extend the observation that salivarian trypanosomes shed surface coat material during the course of infection. Thus, sloughed VSA may be the parasite product that has been hypothesized to cause the nonspecific lymphocyte proliferation, immunosuppression, and/or hypergammaglobulinemia which occur during African trypanosomiasis.