Philippe Vincendeau

l-Arginine Availability Modulates Local Nitric Oxide Production and Parasite Killing in Experimental Trypanosomiasis

ABSTRACT Nitric oxide (NO) is an important effector molecule of the immune system in eliminating numerous pathogens. Peritoneal macrophages fromTrypanosoma brucei brucei-infected mice express type II NO synthase (NOS-II), produce NO, and kill parasites in the presence ofl-arginine in vitro. Nevertheless, parasites proliferate in the vicinity of these macrophages in vivo. The present study shows thatl-arginine availability modulates NO production. Trypanosomes use l-arginine for polyamine synthesis, required for DNA and trypanothione synthesis. Moreover, arginase activity is up-regulated in macrophages from infected mice from the first days of infection. Arginase competes with NOS-II for their common substrate, l-arginine. In vitro, arginase inhibitors decreased urea production, increased macrophage nitrite production, and restored trypanosome killing. In vivo, a dramatic decrease inl-arginine concentration was observed in plasma from infected mice. In situ restoration of NO production and trypanosome killing were observed when excess l-arginine, but notd-arginine or l-arginine plusNω-nitro-l-arginine (a NOS inhibitor), was injected into the peritoneum of infected mice. These data indicate the role of l-arginine depletion, induced by arginase and parasites, in modulating the l-arginine–NO pathway under pathophysiological conditions.

Arginases in parasitic diseases

Abstract Parasites have elaborated a variety of strategies for invading hosts and escaping immune responses. This article proposes that a common mechanism whereby different parasites escape nitric oxide (NO) toxicity is the activation of arginase. This leads to a depletion of l-arginine (substrate of NO synthase, resulting in lower levels of cytotoxic NO) and increased production of polyamines (necessary for parasite growth and differentiation).

Nitric Oxide-Mediated Proteasome-Dependent Oligonucleosomal DNA Fragmentation in Leishmania amazonensis Amastigotes

ABSTRACT Resistance to leishmanial infections depends on intracellular parasite killing by activated host macrophages through the l-arginine-nitric oxide (NO) metabolic pathway. Here we investigate the cell death process induced by NO for the intracellular protozoan Leishmania amazonensis. Exposure of amastigotes to moderate concentrations of NO-donating compounds (acidified sodium nitrite NaNO2 or nitrosylated albumin) or to endogenous NO produced by lipopolysaccharide or gamma interferon treatment of infected macrophages resulted in a dramatic time-dependent cell death. The combined use of several standard DNA status analysis techniques (including electrophoresis ladder banding patterns, YOPRO-1 staining in flow cytofluorometry, and in situ recognition of DNA strand breaks by TUNEL [terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling] assay) revealed a rapid and extensive fragmentation of nuclear DNA in both axenic and intracellular NO-treated amastigotes of L. amazonensis. Despite some similarities to apoptosis, the nuclease activation responsible for characteristic DNA degradation was not under the control of caspase activity as indicated by the lack of involvement of cell-permeable inhibitors of caspases and cysteine proteases. In contrast, exposure of NO-treated amastigotes with specific proteasome inhibitors, such as lactacystin or calpain inhibitor I, markedly reduced the induction of the NO-mediated apoptosis-like process. These data strongly suggest that NO-induced oligonucleosomal DNA fragmentation in Leishmania amastigotes is, at least in part, regulated by noncaspase proteases of the proteasome. The determination of biochemical pathways leading up to cell death might ultimately allow the identification of new therapeutic targets.

Immunology and immunopathology of African trypanosomiasis

Human African trypanosomiasis (HAT) is characterized by a major deregulation of the immune system. Hypergammaglobulinemia, auto-antibodies, and immunodepression are cardinal features. Parasitemia occurs in waves due to the successive appearance of parasites with different variable glycoprotein surface antigens (VGSA). Antigenic variation enables parasites to elude the hosts immune defenses. Although high levels of immune complexes have been detected during HAT, it seems unlikely that they play a significant pathophysiological role. Numerous auto-antibodies have been detected. B lymphocyte activation is uncommon. In vitro T lymphocytes do not proliferate normally, but synthesize cytokines, such as interferon-g which enhance parasite development. Macrophages bind and destroy parasites in the presence of antibodies. They also synthesize large quantities of TNF-alpha which promote parasite destruction but also increase the severity of clinical symptoms. Nitric acid synthesized by activated macrophages has an antiparasitic effect but induces immunosuppression. In the meningoencephalitic stage of HAT, a severe inflammatory reaction is observed. This event is preceded by astroglia which could be induced by astrocytes secreting TNF-a and IL-1. Auto-antibodies against the central nervous system (e.g. anti-galactocerebrosides, anti-tryptophan-like auto-antibodies) may also be involved in the development of encephalitis. VGSA play a key role in the immunopathology of HAT (antigenic variation, induction of cytokine and autoantibody production). Successive relapses occur with the appearance of new antigenic variants and production of antibodies. The resulting continuous stimulation of the immune system leads to deregulation of immunoglobulin production and cytokine network.

Mouse Strain Susceptibility to Trypanosome Infection: An Arginase-Dependent Effect

We previously reported that macrophage arginase inhibits NO-dependent trypanosome killing in vitro and in vivo. BALB/c and C57BL/6 mice are known to be susceptible and resistant to trypanosome infection, respectively. Hence, we assessed the expression and the role of inducible NO synthase (iNOS) and arginase in these two mouse strains infected with Trypanosoma brucei brucei. Arginase I and arginase II mRNA expression was higher in macrophages from infected BALB/c compared with those from C57BL/6 mice, whereas iNOS mRNA was up-regulated at the same level in both phenotypes. Similarly, arginase activity was more important in macrophages from infected BALB/c vs infected C57BL/6 mice. Moreover, increase of arginase I and arginase II mRNA levels and of macrophage arginase activity was directly induced by trypanosomes, with a higher level in BALB/c compared with C57BL/6 mice. Neither iNOS expression nor NO production was stimulated by trypanosomes in vitro. The high level of arginase activity in T. brucei brucei-infected BALB/c macrophages strongly inhibited macrophage NO production, which in turn resulted in less trypanosome killing compared with C57BL/6 macrophages. NO generation and parasite killing were restored to the same level in BALB/c and C57BL/6 macrophages when arginase was specifically inhibited with Nω-hydroxy-nor-l-arginine. In conclusion, host arginase represents a marker of resistance/susceptibility to trypanosome infections.

Quercetin Induces Apoptosis of Trypanosoma brucei gambiense and Decreases the Proinflammatory Response of Human Macrophages

ABSTRACT In addition to parasite spread, the severity of disease observed in cases of human African trypanosomiasis (HAT), or sleeping sickness, is associated with increased levels of inflammatory mediators, including tumor necrosis factor (TNF)-α and nitric oxide derivatives. In the present study, quercetin (3,3′,4′,5,7-pentahydroxyflavone), a potent immunomodulating flavonoid, was shown to directly induce the death of Trypanosoma brucei gambiense, the causative agent of HAT, without affecting normal human cell viability. Quercetin directly promoted T. b. gambiense death by apoptosis as shown by Annexin V binding. In addition to microbicidal activity, quercetin induced dose-dependent decreases in the levels of TNF-α and nitric oxide produced by activated human macrophages. These results highlight the potential use of quercetin as an antimicrobial and anti-inflammatory agent for the treatment of African trypanomiasis.

Atypical human infections by animal trypanosomes

The two classical forms of human trypanosomoses are sleeping sickness due to Trypanosoma brucei gambiense or T. brucei rhodesiense, and Chagas disease due to T. cruzi. However, a number of atypical human infections caused by other T. species (or sub-species) have been reported, namely due to T. brucei brucei, T. vivax, T. congolense, T. evansi, T. lewisi, and T. lewisi-like. These cases are reviewed here. Some infections were transient in nature, while others required treatments that were successful in most cases, although two cases were fatal. A recent case of infection due to T. evansi was related to a lack of apolipoprotein L-I, but T. lewisi infections were not related to immunosuppression or specific human genetic profiles. Out of 19 patients, eight were confirmed between 1974 and 2010, thanks to improved molecular techniques. However, the number of cases of atypical human trypanosomoses might be underestimated. Thus, improvement, evaluation of new diagnostic tests, and field investigations are required for detection and confirmation of these atypical cases.

Human Macrophage Tumor Necrosis Factor (TNF)–α Production Induced by Trypanosoma brucei gambiense and the Role of TNF-α in Parasite Control

Trypanosoma brucei gambiense, a causative agent of sleeping sickness, induced a dose-de- pendent production of tumor necrosis factor (TNF)-a by human macrophages in vitro. TNF- a! was also induced in the Mono Mac 6 cell line, which indicates a direct effect of parasite components on macrophages. Parasite-soluble factors were also potent inducers of TNF-a. The addition of anti-TNF-a to cocultures of macrophages and parasites increased the number of trypanosomes and their life span, whereas irrelevant antibodies had no effect. TNF-o( may have a direct role (i.e., direct trypanolytic activity) andlor an indirect one, such as TNF- a-mediated induction of cytotoxic molecules. A direct dose-dependent lytic effect of TNF-a on purified parasites was observed. This lytic effect was inhibited by anti-TNF-a. These data suggest that, as in experimental trypanosomiasis, TNF-o( is involved in parasite growth control in human African trypanosomiasis. Trypanosoma brucei gainbiense and Trypanosoma brucei rliodesiense, the causative agepts of human African trypano- somiasis (HAT), also called sleeping sickness, are tse-tse fly- transmitted protozoa that multiply extracellularly in the blood- &em, lymph, and interstitial fluids of their hosts (l). There is currently a huge resurgence of HAT because of the deterioration of health facilities, war, and civil disturbances. Blood monocytes and tissue macrophages play a key role in the control of protozoan parasites. Increases in the number of macrophages and the presence of macrophage activation mark- ers are noted in trypanosomiasis (2). Macrophages synthesize effector molecules with antitumoral and antimicrobial prop- erties, including tumor necrosis factor (TNF)-a!. TNF-a! fulfills important functions in host-parasite interactions and plays an important role in controlling infections by various pathogens. TNF-a! is also involved in the pathogenesis of septic shock and systemic inflammatory reactions (3). Trypanosomiasis was one of the first diseases in which the involvement of TNF-a! was observed (3). Trypanosome-derived

Evaluation of FRET real-time PCR assay for rapid detection and differentiation of Plasmodium species in returning travellers and migrants

BackgroundA simple real-time PCR assay using one set of primer and probe for rapid, sensitive and quantitative detection of Plasmodium species, with simultaneous differentiation of Plasmodium falciparum from the three other Plasmodium species (Plasmodium vivax, Plasmodium ovale and Plasmodium malariae) in febrile returning travellers and migrants was developed and evaluated.MethodsConsensus primers were used to amplify a species-specific region of the multicopy 18S rRNA gene, and fluorescence resonance energy transfer hybridization probes were used for detection in a LightCycler platform (Roche). The anchor probe sequence was designed to be perfect matches to the 18S rRNA gene of the fourth Plasmodium species, while the acceptor probe sequence was designed for P. falciparum over a region containing one mismatched, which allowed differentiation of the three other Plasmodium species. The performance characteristics of the real-time PCR assay were compared with those of conventional PCR and microscopy-based diagnosis from 119 individuals with a suspected clinical diagnostic of imported malaria.ResultsBlood samples with parasite densities less than 0.01% were all detected, and analytical sensitivity was 0.5 parasite per PCR reaction. The melt curve means Tms (standard deviation) in clinical isolates were 60.5°C (0.6°C) for P. falciparum infection and 64.6°C (1.8°C) for non-P. falciparum species. These Tms values of the P. falciparum or non-P. falciparum species did not vary with the geographic origin of the parasite. The real-time PCR results correlated with conventional PCR using both genus-specific (Kappa coefficient: 0.95, 95% confidence interval: 0.9 – 1) or P. falciparum-specific (0.91, 0.8 – 1) primers, or with the microscopy results (0.70, 0.6 – 0.8). The real-time assay was 100% sensitive and specific for differentiation of P. falciparum to non-P. falciparum species, compared with conventional PCR or microscopy. The real-time PCR assay can also detect individuals with mixed infections (P. falciparum and non-P. falciparum sp.) in the same sample.ConclusionThis real-time PCR assay with melting curve analysis is rapid, and specific for the detection and differentiation of P. falciparum to other Plasmodium species. The suitability for routine use of this assay in clinical diagnostic laboratories is discussed.

Identification of total and differentially expressed excreted-secreted proteins from Trypanosoma congolense strains exhibiting different virulence and pathogenicity.

Animal trypanosomosis is a major constraint to livestock productivity in the tropics and has a significant impact on the life of millions of people globally (mainly in Africa, South America and south-east Asia). In Africa, the disease in livestock is caused mainly by Trypanosoma congolense, Trypanosoma vivax, Trypanosoma evansi and Trypanosoma brucei brucei. The extracellular position of trypanosomes in the bloodstream of their host requires consideration of both the parasite and its naturally excreted-secreted factors (secretome) in the course of pathophysiological processes. We therefore developed and standardised a method to produce purified proteomes and secretomes of African trypanosomes. In this study, two strains of T. congolense exhibiting opposite properties of both virulence and pathogenicity were further investigated through their secretome expression and its involvement in host-parasite interactions. We used a combined proteomic approach (one-dimensional SDS-PAGE and two-dimensional differential in-gel electrophoresis coupled to mass spectrometry) to characterise the whole and differentially expressed protein contents of secretomes. The molecular identification of differentially expressed trypanosome molecules and their correlation with either the virulence process or pathogenicity are discussed with regard to their potential as new diagnostic or therapeutic tools against animal trypanosomosis.