Nitric oxide-mediated cytostatic activity on Trypanosoma brucei gambiense and Trypanosoma brucei brucei.

Macrophages collected from BCG-infected mice or exposed in vitro to interferon-gamma plus lipopolysaccharide developed a cytostatic activity on Trypanosoma brucei gambiense and Trypanosoma brucei brucei. This trypanostatic activity of activated macrophages was inhibited by addition of N-monomethyl-L-arginine, an inhibitor of the L-arginine-nitric oxide (NO) metabolic pathway, indicating a role for NO as the effector molecule. Contrary to trypanosomes treated with N2gas, trypanosomes treated with NO gas did not proliferate in vitro on normal macrophages. Compared to mice infected with control parasites, mice infected with NO-treated parasites had decreased parasitemias in the first days postinfection and had a prolonged survival. Addition of excess iron reversed the trypanostatic effect of both activated macrophages and NO gas. These data show that activated macrophages exert an antimicrobial effect on T.b. gambiense and T.b. brucei through the L-arginine-NO metabolic pathway. In trypanosomes, NO could trigger iron loss from critical targets involved in parasite division. The participation of this effector mechanism among the other immune elements involved in the control of African trypanosomes (antibodies, complement, phagocytic events) remains to be defined.     

Effects of anti-protozoal drugs and histopathological studies on trypanosome species

The trypanosomostatic and trypanosomicidal effects of four anti-protozoal drugs, namely halofantrine hydrochloride, chloroquine phosphate, benzoylmetronidazole and pyrimethamine, on species of trypanosomes, viz. Trypanosoma brucei brucei (MBOS/NG/94/NITR) Bassa strain, T. congolense (MBOS/NG/93/NVRI) Zaria strain and T. brucei gambiense (MHOM/NG/92/NITR) Abraka strain, were investigated. In vitro and in vivo studies on these drugs vis-a-vis the parasites were carried out. The histopathological changes in organs and tissues of experimentally infected rats were also studied. Results from the in vitro studies indicated that halofantrine hydrochloride, chloroquine phosphate, benzoylmetronidazole and pyrimethamine appeared to be effective trypanosomicidal agents against T. brucei brucei (Bassa strain), T. congolense (Zaria strain) and T. brucei gambiense (Abraka strain). The in vivo studies showed that these drugs were sub-curative by prolonging the survival period of the trypanosome-infected rats, but not necessarily curing the infection. Histopathological findings indicated inflammatory reactions characterised by infiltration to variable degrees in the majority of tissues, mostly in the lungs and liver. The most consistent lesions were interstitial pneumonia, multifocal necrosis and oedema. Pathological findings showed the T. brucei brucei and T. brucei gambiense strains studied to be both intravascular and extravascular parasites. These results suggest that halofantrine hydrochloride, chloroquine phosphate, benzoylmetronidazole and pyrimethamine could be used as supportive, suppressive and/or synergistic/additive drugs in the treatment of African trypanosomiasis. Their effects on species of trypanosomes have been studied and are reported for the first time.     

Differential effects of interferon-gamma on production of trypanosome-derived lymphocyte-triggering factor by Trypanosoma brucei gambiense and Trypanosoma brucei brucei

Trypanosome-derived lymphocyte-triggering factor (TLTF) produced by Trypanosoma brucei brucei stimulates production of interferon-gamma (IFN-gamma) by CD8+ T cells, and it is reported that, in turn, IFN-gamma stimulates proliferation of T. b. brucei. We studied the role of TLTF in trypanosome proliferation using the Wellcome strain (WS) of Trypanosoma brucei gambiense and the ILtat 1.4 strain (IL) of T. b. brucei. Increase in the number of WS in infected rats is more rapid than IL and corresponds with comparatively higher levels of IFN-gamma. Production of IFN-gamma, as measured by protein and messenger RNA (mRNA) levels, was maintained by splenocytes from WS-infected rats, whereas levels decreased in IL-infected rats, accompanied by prolongation of infection. Expression of TLTF mRNA by in vitro-cultured WS was promoted in a dose-dependent fashion by addition of recombinant rat IFN-gamma at all concentrations tested. The addition of lower concentrations of IFN-gamma to cultured IL increased expression of TLTF mRNA, whereas, in contrast to WS, addition of 100 and 1,000 U/ml IFN-gamma decreased expression of TLTF by IL. These results show that unlike WS, elevated IFN-gamma concentrations lead to decreased TLTF production by IL. It is believed that decreased TLTF production in IL-infected rats leads to lowered IFN-gamma production, thereby slowing IL proliferation.     

Experimental infections of laboratory rodents with recently isolated stocks of Trypanosoma brucei gambiense. 1. Parasitological investigations

Albino rats and white mice were infected with populations of Trypanosoma brucei gambiense isolated from patients in four different areas in Central Africa. Differences in virulence as shown by the level of parasitaemia, number of relapses, and length of survival time, were observed amongst the stocks according to their geographical origin and secondarily to the degree of adaptation to the rodents. All the stocks are pathogenic for the laboratory rodents, and the presence of extravascular trypanosomes in the brain was confirmed in all infected animals. Spleen, liver, and kidneys were less constantly found to be positive. The morphology of the extravascular parasites was highly variable, ranging from long slender trypomastigotes to spheromastigotes and even amastigotes.     

Effects of polyamines on two strains of Trypanosoma brucei in infected rats and in vitro culture

We studied the effects of polyamines, which are necessary for proliferation and antioxidation in Trypanosoma brucei gambiense Wellcome strain (WS) and Trypanosoma brucei brucei ILtat 1.4 strain (IL). No difference was found in activity of ornithine decarboxylase (ODC), a key enzyme in polyamine synthesis in trypanosomes, in both strains maintained in vitro; higher (P < 0.05) ODC values were found in IL in vivo. However, WS in vivo exhibited higher proliferation rates with higher spermidine content and decreased host survival times than IL. The in vitro proliferation and polyamine contents of WS increased with the addition of polyamine to the 1-difluoromethylornithine culture medium, but not IL. These results suggested that WS uses extracellular polyamine for proliferation. In the in vitro culture, WS was less tolerant of hydrogen peroxide (oxidative stress) than IL, and malondialdehyde levels in WS were higher than in IL. The expression of trypanothione synthetase mRNA in WS in vitro was higher than in IL. These results suggest that IL is dependent on the synthesis of polyamines for proliferation and reduction of oxidative stress, whereas WS is dependent on the uptake of extracellular polyamines. A thorough understanding of the differences in the metabolic capabilities of various trypanosomes is important for the design of more effective medical treatments.     

Evidence for a Trypanosoma brucei lipoprotein scavenger receptor

African trypanosomes are lipid auxotrophs that live in the bloodstream of their human and animal hosts. Trypanosomes require lipoproteins in addition to other serum components in order to multiply under axenic culture conditions. Delipidation of the lipoproteins abrogates their capacity to support trypanosome growth. Both major classes of serum lipoproteins, LDL and HDL, are primary sources of lipids, delivering cholesterol esters, cholesterol, and phospholipids to trypanosomes. We show evidence for the existence of a trypanosome lipoprotein scavenger receptor, which facilitates the endocytosis of both native and modified lipoproteins, including HDL and LDL. This lipoprotein scavenger receptor also exhibits selective lipid uptake, whereby the uptake of the lipid components of the lipoprotein exceeds that of the protein components. Trypanosome lytic factor (TLF1), an unusual HDL found in human serum that protects from infection by lysing Trypanosoma brucei brucei, is also bound and endocytosed by this lipoprotein scavenger receptor. HDL and LDL compete for the binding and uptake of TLF1 and thereby attenuate the trypanosome lysis mediated by TLF1. We also show that a mammalian scavenger receptor facilitates lipid uptake from TLF1 in a manner similar to the trypanosome scavenger receptor. Based on these results we propose that HDL, LDL, and TLF1 are all bound and taken up by a lipoprotein scavenger receptor, which may constitute the parasite's major pathway mediating the uptake of essential lipids.     

Procyclic tsetse fly midgut forms and culture forms of African trypanosomes share stage- and species-specific surface antigens identified by monoclonal antibodies

Procyclic culture form (PCF) trypanosomes were established from a bloodstream form population of cloned Trypanosoma brucei rhodesiense and were used to immunize mice for hybridoma production. Indirect immunofluorescence was used to select 10 hybridomas which secreted antibodies that bound to the surface of homologous living PCF. The antibodies reacted with PCF of several clones of T.b. brucei, T.b. gambiense, and T.b. rhodesiense, but not with PCF of T. congolense or T. vivax, or with promastigotes of several species of Leishmania parasites. The antigens were not detectable in ethanol/acetic acid-fixed bloodstream forms or in lysates of bloodstream forms of any of the T. brucei subspecies, and are thus species-specific and stage-specific markers. Selected monoclonal antibodies bound to procyclic trypanosomes taken directly from the midgut of infected tsetse flies, and to immature epimastigote forms in salivary probes, and may therefore be useful in epidemiologic investigations.     

Vector competence of Glossina palpalis gambiensis for Trypanosoma brucei s.l. and genetic diversity of the symbiont Sodalis glossinidius

Tsetse flies transmit African trypanosomes, responsible for sleeping sickness in humans and nagana in animals. This disease affects many people with considerable impact on public health and economy in sub-Saharan Africa, whereas trypanosomes' resistance to drugs is rising. The symbiont Sodalis glossinidius is considered to play a role in the ability of the fly to acquire trypanosomes. Different species of Glossina were shown to harbor genetically distinct populations of S. glossinidius. We therefore investigated whether vector competence for a given trypanosome species could be linked to the presence of specific genotypes of S. glossinidius. Glossina palpalis gambiensis individuals were fed on blood infected either with Trypanosoma brucei gambiense or Trypanosoma brucei brucei. The genetic diversity of S. glossinidius strains isolated from infected and noninfected dissected flies was investigated using amplified fragment length polymorphism markers. Correspondence between occurrence of these markers and parasite establishment was analyzed using multivariate analysis. Sodalis glossinidius strains isolated from T. brucei gambiense-infected flies clustered differently than that isolated from T. brucei brucei-infected individuals. The ability of T. brucei gambiense and T. brucei brucei to establish in G. palpalis gambiensis insect midgut is statistically linked to the presence of specific genotypes of S. glossinidius. This could explain variations in Glossina vector competence in the wild. Then, assessment of the prevalence of specific S. glossinidius genotypes could lead to novel risk management strategies.     

Tsetse-trypanosome interactions: rites of passage.

Trypanosomes that cause sleeping sickness (Trypanosoma brucei rhodesiense and T. b. gambiense) are entirely dependent on tsetse for their transmission between hosts, but the flies are not easily infected. This situation has not arisen by chance - the tsetse has evolved an efficient defence system against trypanosome invasion. In this review, Susan Welburn and Ian Maudlin chart the progress of trypanosomes through the fly and identify some of the hazards faced by both parasite and fly that affect vector competence of tsetse.     

Experimental evaluation of xenodiagnosis to detect trypanosomes at low parasitaemia levels in infected hosts

In Human African Trypanosomosis (HAT) endemic areas, there are a number of subjects that are positive to serological tests but in whom trypanosomes are difficult to detect with the available parasitological tests. In most cases and particularly in West Africa, these subjects remain untreated, thus posing a fundamental problem both at the individual level (because of a possible lethal evolution of the disease) and at the epidemiological level (since they are potential reservoirs of trypanosomes). Xenodiagnosis may constitute an alternative for this type of cases. The objective of this study was to update the use of xenodiagnosis to detect trypanosomes in infected host characterized by low parasitaemia levels. This was carried out experimentally by infecting cattle and pigs with Trypanosoma congolense and T. brucei gambiense respectively, and by feeding tsetse flies (Glossina morsitans submorsitans and G. palpalis gambiensis, from the CIRDES colonies) on these animals at a time when the observed blood parasitaemia were low or undetectable by the classical microscopic parasitological tests used for the monitoring of infected animals. Our results showed that: i) the G. p. gambiensis colony at CIRDES could not be infected with the T. b. gambiense stocks used; ii) midgut infections of G. m. submorsitans were observed with both T. congolense and T. b. gambiense; iii) xenodiagnosis remains positive even at very low blood parasitaemia for both T. congolense and T. b. gambiense; and iv) to implement T. b. gambiense xenodiagnosis, batches of 20 G. m. submorsitans should be dissected two days after the infective meal. These results constitute a first step toward a possible implementation of xenodiagnosis to better characterize the parasitological status of seropositive individuals and the modalities of parasite transmission in HAT foci.     

African trypanosome interactions with an in vitro model of the human blood-brain barrier

The neurological manifestations of sleeping sickness in man are attributed to the penetration of the blood-brain barrier (BBB) and invasion of the central nervous system by Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. However, how African trypanosomes cross the BBB remains an unresolved issue. We have examined the traversal of African trypanosomes across the human BBB using an in vitro BBB model system constructed of human brain microvascular endothelial cells (BMECs) grown on Costar Transwell inserts. Human-infective T. b. gambiense strain IL 1852 was found to cross human BMECs far more readily than the animal-infective Trypanosoma brucei brucei strains 427 and TREU 927. Tsetse fly-infective procyclic trypomastigotes did not cross the human BMECs either alone or when coincubated with bloodstreamform T. b. gambiense. After overnight incubation, the integrity of the human BMEC monolayer measured by transendothelial electrical resistance was maintained on the inserts relative to the controls when the endothelial cells were incubated with T. b. brucei. However, decreases in electrical resistance were observed when the BMEC-coated inserts were incubated with T. b. gambiense. Light and electron microscopy studies revealed that T. b. gambiense initially bind at or near intercellular junctions before crossing the BBB paracellularly. This is the first demonstration of paracellular traversal of African trypanosomes across the BBB. Further studies are required to determine the mechanism of BBB traversal by these parasites at the cellular and molecular level.     

Haptoglobin-related protein mediates trypanosome lytic factor binding to trypanosomes

Trypanosome lytic factor (TLF-1) is an unusual high density lipoprotein (HDL) found in human serum that is toxic to Trypanosoma brucei brucei and may be critical in preventing human infections by this parasite. TLF-1 is composed of four major apolipoproteins: apolipoprotein AI, apolipoprotein AII, paraoxonase, and the primate-specific haptoglobin-related protein (Hpr). Hpr is greater than 90% homologous to haptoglobin (Hp), an abundant acute phase serum protein. Killing of trypanosomes by TLF-1 requires cell surface binding, endocytosis, and subsequent lysosomal targeting. Low temperature binding studies reveal two receptors for TLF-1: one that is high affinity/low capacity (K(d) approximately 12 nm, 350 receptors per cell) and another that binds with low affinity/high capacity (K(d) approximately 1 microm, 60,000 receptors per cell). The low affinity binding is competed by nonlytic human HDL and is likely to be apolipoprotein AI-mediated. Purified human Hpr and human Hp bind to trypanosomes, are internalized, and are targeted to the lysosome. Furthermore, Hpr shows competition for TLF-1 binding, and a monoclonal antibody against Hpr prevents both TLF-1 uptake and trypanosome killing. Based on these results, we propose that Hpr mediates the high affinity binding of TLF-1 to T. b. brucei through a haptoglobin-like receptor.     

Will the real Trypanosoma brucei rhodesiense please step forward?

The sleeping sickness trypanosomes Trypanosoma brucei rhodesiense and T. brucei gambiense are morphologically indistinguishable from each other and from T. brucei brucei, which does not infect humans. The relationships between these three subspecies have been controversial. Several years ago, the characterization of T. brucei gambiense was reviewed in an attempt to clarify and draw together the results, and to put them in the context of the biology of the organism. The discovery of a gene associated with human-serum resistance in T. brucei rhodesiense and the consequent reappraisal of the identity of this trypanosome prompt this companion article.     

Detection of nitrosylated epitopes in Trypanosoma brucei gambiense by polyclonal and monoclonal anti-conjugated-NO-cysteine antibodies

Activated macrophages with the Calmette/Guérin bacillus (BCG) have a cytotoxic/cytostatic effect on the extracellular parasite, Trypanosoma brucei gambiense. This effect was inhibited when the NO-synthase inhibitor NG-monomethyl-L-arginine (NMMA; 0.5 mM) was added to the culture media. Using an immunocytochemical method with rabbit polyclonal or mouse monoclonal antibodies directed against conjugated nitroso-epitopes (anti-conjugated-NO-cysteine), nitrosylated antigens were visualized in fixed trypanosomes. These results suggest that NO was synthesized by the activated macrophages and that it reacted with some parasitic proteins containing cysteine. The release of NO bound to parasitic proteins may cause the killing of trypanosomes. The immunoreactivity was positive when the trypanosomes were obtained from the supernatant of the BCG-activated macrophages that contains BSA (4 mg/mL). In contrast, the parasites cocultured with non-activated macrophages remained completely viable, and, the immunoreactivity was completely negative.     

Trypanosoma brucei: infectivity and immunogenicity of cultured parasites

Trypanosoma brucei brucei, derived from the salivary glands of infected tsetse flies (Glossina morsitans morsitans) and maintained in culture for over 4 years, were infective to both albino rats and tsetse flies. Virulence was markedly enhanced during the first passage in albino rats or tsetse flies. Irradiated cultured trypanosomes induced immunity to homologous challenge but not to tsetse fly or blood-induced challenge with the same stock.     

Trypanosoma brucei gambiense: growth in vitro of bloodstream forms inhibited by dichlorodiammineplatinum (II) compounds and hypolipidemic drugs

Infectious bloodstream forms of Trypanosoma brucei gambiense were grown in microcultures of murine bone marrow cells in 96-well tissue culture plates. Limiting dilution studies showed that fewer than 10 cultured trypanosomes developed into populations of about 5 X 10(4) parasites per well in a week. Bloodstream parasites were reisolated with high efficiency from mice infected with cultured parasites; fewer than 10 bloodstream parasites successfully established a trypanosome population in a microculture. Both the cis and trans isomers of dichlorodiammineplatinum (II) (cisplatin and transplatin) and a hypolipidemic agent, Wy 14643, were found to have activity against T. b. gambiense growing in microcultures. The minimum concentration of drug necessary to completely eliminate parasites from microcultures was 4 microM for cisplatin, 40 microM for transplatin, and 500 microM for Wy 14643. A preformed complex of cisplatin and bovine serum albumin and another hypolipidemic agent, chlofibric acid, were inactive. This culture system should be useful for rapid screening of large numbers of compounds for trypanocidal activity.     

Human high density lipoproteins are platforms for the assembly of multi-component innate immune complexes

Human innate immunity to non-pathogenic species of African trypanosomes is provided by human high density lipoprotein (HDL) particles. Here we show that native human HDLs containing haptoglobin-related protein (Hpr), apolipoprotein L-I (apoL-I) and apolipoprotein A-I (apoA-I) are the principle antimicrobial molecules providing protection from trypanosome infection. Other HDL subclasses containing either apoA-I and apoL-I or apoA-I and Hpr have reduced trypanolytic activity, whereas HDL subclasses lacking apoL-I and Hpr are non-toxic to trypanosomes. Highly purified, lipid-free Hpr and apoL-I were both toxic to Trypanosoma brucei brucei but with specific activities at least 500-fold less than those of native HDLs, suggesting that association of these apolipoproteins within the HDL particle was necessary for optimal cytotoxicity. These studies show that HDLs can serve as platforms for the assembly of multiple synergistic proteins and that these assemblies may play a critical role in the evolution of primate-specific innate immunity to trypanosome infection.     

Entry of Trypanosoma brucei gambiense into microvascular endothelial cells of the human blood-brain barrier

Using an in vitro model of the human blood-brain barrier consisting of human brain microvascular endothelial cells we recently demonstrated that Trypanosoma brucei gambiense bloodstream-forms efficiently cross these cells via a paracellular route while Trypanosoma brucei brucei crosses these cells poorly. Using a combination of techniques that include fluorescence activated cell sorting, confocal and electron microscopy, we now show that some T.b. gambiense blood stream form parasites have the capacity to enter human brain microvascular endothelial cells. The intracellular location of the trypanosomes was demonstrated in relation to the endothelial cell plasma membrane and to the actin cytoskeleton. These parasites may be a terminal stage within a lysosomal compartment or they may be viable trypanosomes that will be able to exit the brain microvascular endothelial cells. This process may provide an additional transcellular route by which the parasites cross the blood-brain barrier.     

Cultivation in a semi-defined medium of animal infective forms of Trypanosoma brucei, T. equiperdum, T. evansi, T. rhodesiense and T. gambiense.

A semi-defined medium for the cultivation of bloodstream forms of the African trypanosome brucei subgroup was developed. Out of 14 different strains tested, 10 could be cultured including Trypanosoma brucei, T. equiperdum, T. evansi, T. rhodesiense and T. gambiense. The presence of a reducing agent (2-mercaptoethanol or thioglycerol) was found to be essential for growth. The standard medium consisted of Hepes buffered minimum essential medium with Earle's salts supplemented with 0.2 mM 2-mercaptoethanol, 2 mM pyruvate and 10% inactivated serum either from rabbit (T. brucei, T. equiperdum, T. evansi and T. rhodesiense) or human (T. gambiense). Although a general medium could be defined for the long-term maintenance of trypanosome cultures, the initiation to culture nevertheless required particular conditions for the different strains. The cultured trypanosomes had all the characteristics of the in vivo bloodstream forms including: morphology, infectivity, antigenic variation and glucose metabolism.