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.     

Complement reversal of immunosuppression induced with plasma of rats infected with Trypanosoma brucei rhodesiense

Suppression of antibody production by splenic lymphocytes from rats immunized with sheep red blood cells (SRBC) after incubation with plasma from rats infected with Trypanosoma brucei rhodesiense was confirmed. Suppressive activity became evident in plasma after the sixth day of infection and was manifested by reduction in the number of hemolytic Jerne plaques produced by the treated cells. The activity was temporally associated with increased amounts of soluble immune complex (SIC) reduced titers of lytic complement, elevated titers of immunoconglutinin (IK) and anemia. Treatment of suppressive plasma with hemolysin sensitized SRBC alexinated with horse complement to reduce IK did not reduce suppressive activity, and the activity appeared to have been enhanced when the plasma was heated to inactivate the remaining complement (C'). When fresh rat C' was added to the treated cells, the suppression was largely, though not completely, reversed. Treatment of spleen cells with SIC prepared in vitro from bovine serum albumin (BSA) and rabbit antiBSA also suppressed the plaque forming capacity of the cells. Complexes of BSA-antiBSA-C' and complexes of BSA-antiBSA-C'-IK were equally suppressive. Again, addition of fresh C' to cells treated with these complexes largely, though not completely, reversed the suppressive effect on the cells. From the results it is suggested that immunosuppression associated with experimental T. b. rhodesiense infection may be in part a suppression of the capacity of induced lymphocytes to produce antibody. It is possible that the suppression was mediated by SIC present in the plasma of the infected rats and this effect was probably enhanced by reduced levels of complement in the suppressive plasma.     

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.     

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.     

Enhanced endothelial nitric oxide-synthase activity in mice infected with Trypanosoma brucei

Infection of humans with Trypanosoma brucei causes sleeping sickness, which is invariably fatal if left untreated. The course of infection is characterised, among others, by multiple organ damage including cardiovascular dysfunctions such as hypotension and breakdown of the blood-brain barrier. The latter eventually leads to the parasite invasion into central nervous system and ultimately to the death of the patient. Nitric oxide (NO) synthesised from L-arginine via endothelial NO-synthase (eNOS) is involved in the control of vascular tone and permeability. The present study explores the effect of T. brucei infection on the endothelium-dependent in vitro vasomotor response of isolated mouse aortas. Aorta rings were suspended in organ chambers for isometric tension recording. The endothelium-dependent NO-mediated relaxation in response to acetylcholine (10(-9) to 10(-5) M) was markedly enhanced in the infected mice compared to controls (P<0.05), whereas the endothelium-independent vasodilation to an exogenous NO-donor, sodium nitroprusside, was comparable in both groups. Norepinephrine-stimulated contraction was also comparable in the absence or presence of the NO-synthase inhibitor N(omega)-Nitro-L-arginine methyl ester (L-NAME; 10(-4)M) in both groups. The enhanced endothelium-dependent relaxation in the infected mice correlated well with a 3.5-fold increase in eNOS protein level in these aortas as compared to those of control mice (P=0.05). Thus, T. brucei infection enhances eNOS protein expression in the endothelium, causing a pronounced vasodilation. Overproduction of NO in trypanosomiasis may be involved in the observed generalised hypotension and in an increased vascular permeability that facilitates T. brucei invasion into surrounding tissues and its penetration into the central nervous system in later phases of infection.     

Delayed expulsion of Hymenolepis diminuta in Trypanosoma brucei infected mice

An experiment was conducted to study the effect of Trypanosoma brucei on the expulsion of Hymenolepis diminuta in the mouse. The study showed that T. brucei given 8 days before infection with H. diminuta resulted in a significant delay in the expulsion of the H. diminuta worm burden. This finding is suggested to be due to the immunosuppressive effect of the trypanosome.     

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.     

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.     

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.     

Trypanosoma brucei: attenuation by cortcosteroids of the anemia of infected mice

BALBc/J mice infected with pleomorphic stabilate of Trypanosoma brucei develop a severe anemia. The anemia is initially normoblastic and normocytic but becomes macrocytic due to brisk and sustained reticulocytosis. Red blood cells from uninfected animals are destroyed when transfused into infected animals. Neither extensive intravascular hemolysis nor red cell agglutinins were detected in infected animals, but spherocytes were common in the peripheral circulation. Treatment of mice with either dexamethasone or hydrocortisone greatly retarded the development of the anemia. Comparison of the course of infection in these treated mice to that in normal mice suggests that these corticosteroids attenuate the anemia through their immunosuppressive action.     

MS and NMR analysis of the cross-reacting determinant glycan from Trypanosoma brucei brucei MITat 1.6 variant specific glycoprotein

The cross-reacting determinant glycan from Trypanosoma brucei brucei MITat 1.6 is known to contain galactose, mannose and non-acetylated glucosamine. The structural elucidation of this oligosaccharide has been impeded by an unusual non-glycosidic linkage to the peptide chain and a glycosidic linkage to inositol phosphate on either side of the oligosaccharide. Using two different approaches for the isolation of the glycan, namely hydrolysis to give the oligosaccharide directly or pronase digestion to yield the glycan-containing C-terminal glycophosphopeptide, the structure of this glycan was elucidated by mass spectrometry and 1H-NMR spectroscopy. There was evidence of heterogeneity in the glycan residue.     

Prolyl oligopeptidase of Trypanosoma brucei hydrolyzes native collagen,peptide hormones and is active in the plasma of infected mice

Proteases play important roles in many biological processes of parasites, including their host interactions. In sleeping sickness, Trypanosoma brucei proteases released into the host bloodstream could hydrolyze host factors, such as hormones, contributing to the development of the disease's symptoms. In this study, we present the identification of the T. brucei prolyl oligopeptidase gene (poptb) and the characterization of its corresponding enzyme, POP Tb. Secondary structure predictions of POP Tb show a structural composition highly similar to other POPs. Recombinant POP Tb produced in E. coli was active and highly sensitive to inhibitors of Trypanosoma cruzi POP Tc80. These inhibitors, which prevent T. cruzi entry into non-phagocytic cells, arrested growth of the T. brucei bloodstream form in a dose-dependent manner. POP Tb hydrolyzes peptide hormones containing Pro or Ala at the P1 position at a slightly alkaline pH, and also cleaves type I collagen in vitro and native collagen present in rat mesentery. Furthermore, POP Tb is released into the bloodstream of T. brucei infected mice where it remains active. These data suggest that POP Tb might contribute to the pathogenesis of sleeping sickness.     

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.     

Lectin interactions with the variant surface glycoprotein from Trypanosoma brucei brucei incorporated into liposomes

The variant specific surface glycoprotein from Trypanosoma brucei brucei is incorporated into lipid vesicles using 8M urea as an unfolding reagent. Pronase treatment of these proteoliposomes removes most of the protein, leaving a glycophospholipopeptide which is the membrane attachment site. We show here that lectins, specific for mannose and galactose are able to recognize oligosaccharide residues on these proteoliposomes, using a straightforward aggregation assay. The relevance of these results obtained with the liposome model system to the accessibility of the surface antigens in living trypanosomes is discussed.     

Trypanosoma brucei brucei: variability in the association of some variant surface glycoproteins

In our isolation procedure, the surface antigens of the variants AnTat 1.1 and 1.10 (Trypanosoma brucei brucei) are essentially obtained as a disulfide-linked dimer while the AnTat 1.8 surface antigen is found as a mixture of monomer and disulfide-linked dimer. This observation may be related to the localization of the cysteine residues in the protein sequences. In the purification procedure using concanavalin-A Sepharose chromatography, besides the VSG elution by methyl-alpha-D-mannopyranoside, a quantitative elution of still bound VSG may be obtained by the addition of beta-mercaptoethanol to methyl-alpha-D-mannopyrannoside in the elution buffer. The surface antigen of the variant AnTat 1.1 was examined for molecular form at several different times during the release procedure. The disulfide-linked dimer could be observed within 30 min of the surface coat release, indicating its presence within the parasite.     

Biosynthesis and processing of a variant surface glycoprotein from Trypanosoma brucei brucei

Iowa trypanosome antigen type (IaTat) 1.2 variant surface glycoprotein (VSG) is synthesized in vitro as a Mr 54,000 preprotein that contains a 31-amino-acid signal peptide. Translation of mRNA in the presence of either dog pancreas or trypanosome microsomal membranes results in cotranslational cleavage of the signal peptide and addition of core oligosaccharide side chains to the protein. Analysis of these products on sodium dodecyl sulfate (SDS)-gels indicates that removal of the signal peptide (Mr 3200) is almost exactly compensated for by an increase in molecular weight due to carbohydrate addition. Pulse-chase experiments in cultures of isolated trypanosomes indicate that two IaTat 1.2 VSG species (Mr 58,000 and 60,000) occur in vivo. When glycosylation is inhibited by incubation of trypanosomes with tunicamycin, a single Mr 50,000 polypeptide is immunoprecipitated. The multiple protein species, therefore, arise from heterogeneity in carbohydrate side chains whose synthesis and transfer to the protein are tunicamycin sensitive. Sequence analysis verified that both species of VSG contain identical amino-terminal sequences. Further post-translational processing of IaTat 1.2 VSG includes addition of phosphate and myristic acid residues, both of which have been shown to be located in the immunologically cross-reactive determinant at the carboxyl terminus of the protein. Exposure of this attachment site requires post-translational proteolytic removal of a 17-amino-acid peptide from the carboxyl terminus of an intermediate form of VSG.     

Trypanosoma brucei brucei: A comparison of gene expression in the liver and spleen of infected mice utilizing cDNA microarray technology

Trypanosoma brucei brucei, the infectious agent of the disease known as Nagana, is a pathogenic trypanosome occurring in Africa, where it causes significant economic loss to domesticated livestock. Although many studies on the histopathology of organs of mice infected with T. b. brucei have been reported, little work has been done regarding gene expression in these organs in infected mice. In this paper, we describe the use of cDNA microarray to determine gene expression profiles in the liver and spleen of mice infected with T. b. brucei (STIB 920) at peak parasitaemia (12 days after infection). Our results showed that a total of 123 genes in the liver and 389 genes in the spleen were expressed differentially in T. b. brucei infected mice. In contrast, however, in an acute infection in mice caused by Trypanosoma brucei evansi, a species genetically related to T. b. brucei, 336 genes in the liver and 190 genes in the spleen were expressed, differentially, indicating that the liver of mice was more affected by the acute T. b. evansi infection whilst the spleen was more affected by the subacute T. b. brucei infection. Our results provide a number of possible reasons why mice infected with T. b. evansi die sooner than those infected with T. b. brucei: (1) mice infected with T. b. evansi may need more stress response proteins to help them pass through the infection and these are probably excessively consumed; (2) proliferating cell nuclear antigen was more down-regulated in the liver of mice infected with T. b. evansi, which indicated that the inhibition of proliferation of hepatocytes in mice infected with T. b. evansi might be more severe than that in T. b. brucei infection; and (3) more hepatocyte apoptosis occurred in the mice infected with T. b. evansi and this might be probably the most important reason why mice died sooner than those infected with T. b. brucei. Studies of the changes in the gene expression profile in the liver and spleen of mice infected with T. b. brucei may be helpful in understanding the mechanisms of pathogenesis in Nagana disease at the molecular level. By comparing the gene profiles of the liver and spleen of mice infected with T. b. brucei with T. b. evansi, we have identified a number of factors that could explain the differences in pathogenesis in mice infected with these two African trypanosomes.