Identification of a tryptophan-like epitope borne by the variable surface glycoprotein (VSG) of African trypanosomes.

Antibodies (Ab) directed against a tryptophan-like epitope (WE) were previously detected in patients with human African trypanosomiasis (HAT). We investigated whether or not these Ab resulted from immunization against trypanosome antigen(s) expressing a WE. By Western blotting, we identified an antigen having an apparent molecular weight ranging from 60 to 65 kDa, recognized by purified rabbit anti-WE Ab. This antigen, present in trypomastigote forms, was absent in procyclic forms and Trypanosoma cruzi trypomastigotes. Using purified variable surface glycoproteins (VSG) from various trypanosomes, we showed that VSG was the parasite antigen recognized by these rabbit Ab. Anti-WE and anti-VSG Ab were purified from HAT sera by affinity chromatography. Immunoreactivity of purified antibodies eluted from affinity columns and of depleted fractions showed that WE was one of the epitopes borne by VSG. These data underline the existence of an invariant WE in the structure of VSG from several species of African trypanosomes.

Phenotypical characteristics, biochemical pathways, molecular targets and putative role of nitric oxide-mediated programmed cell death in Leishmania.

Nitric oxide (NO) has been demonstrated to be the principal effector molecule mediating intracellular killing of Leishmania, both in vitro and in vivo. We investigated the type of cell death process induced by NO for the intracellular amastigote stage of the protozoa Leishmania. Specific detection methods revealed a rapid and extensive cell death with morphological features of apoptosis in axenic amastigotes exposed to NO donors, in intracellular amastigotes inside in vitro - activated mouse macrophages and also in activated macrophages of regressive lesions in a leishmaniasis-resistant mouse model. We extended our investigations to the dog, a natural host-reservoir of Leishmania parasites, by demonstrating that co-incubation of infected macrophages with autologous lymphocytes derived from dogs immunised with purified excreted-secreted antigens of Leishmania resulted in a significant NO-mediated apoptotic cell death of intracellular amastigotes. From the biochemical point of view, NO-mediated Leishmania amastigotes apoptosis did not seem to be controlled by caspase activity as indicated by the lack of effect of cell permeable inhibitors of caspases and cysteine proteases, in contrast to specific proteasome inhibitors, such as lactacystin or calpain inhibitor I. Moreover, addition of the products of two NO molecular targets, cis-aconitase and glyceraldehyde-3-phosphate dehydrogenase, also had an inhibitory effect on the cell death induced by NO. Interestingly, activities of these two enzymes plus 6-phosphogluconate dehydrogenase, parasitic enzymes involved in both glycolysis and respiration processes, are overexpressed in amastigotes selected for their NO resistance. This review focuses on cell death of the intracellular stage of the pathogen Leishmania induced by nitrogen oxides and gives particular attention to the biochemical pathways and the molecular targets potentially involved. Questions about the role of Leishmania amastigotes NO-mediated apoptosis in the overall infection process are raised and discussed.

Lower nitric oxide susceptibility of trivalent antimony-resistant amastigotes of Leishmania infantum.

We previously documented the induction of Leishmania amastigote apoptosis by trivalent antimony (SbIII) and nitric oxide (NO). We demonstrate here that SbIII-resistant amastigotes were resistant to NO toxicity when delivered extracellularly by NO donors or intracellularly via macrophage activation. Shared biochemical targets for SbIII and NO resistance in Leishmania are discussed.

Lymphocytes of dogs immunised with purified excreted-secreted antigens of Leishmania infantum co-incubated with Leishmania infected macrophages produce IFN gamma resulting in nitric oxide-mediated amastigote apoptosis.

The role of nitric oxide (NO) in the anti-leishmanial activity has been confirmed both in vitro and in vivo. Recently, we demonstrated that NO-mediated apoptosis-like amastigote death pathway is an important and highly regulated mechanism used for the clearance of Leishmania within infected murine macrophages stimulated to produce NO endogenously. To further characterize these important effector mechanisms in dog, a natural host-reservoir of L. infantum/L. chagasi, we have developed an ex vivo infection model of canine macrophages. Exposure of L. infantum-infected macrophages to autologous peripheral lymphocytes derived from dogs immunised with purified excreted-secreted antigens of L. infantum promastigotes (LiESAp) formulated with muramyl dipeptide (MDP) as adjuvant resulted in a significant leishmanicidal effect due to interferon (IFN)-gamma dependent macrophage activation. Concomitant accumulation of NO(3)(-)/NO(2)(-) in supernatants of co-cultured cells and in situ staining of parasites with terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) and YOPRO-1 showed that NO-mediated apoptosis of intracellular L. infantum amastigotes is occurring in canine macrophages as previously observed in mouse models. Monitoring these parameters in dogs after immunisation and before experimental challenge can represent a useful and easy way to rapidly evaluate vaccine candidates against canine visceral leishmaniasis.

Antibodies directed against nitrosylated neoepitopes in sera of patients with human African trypanosomiasis.

Antibodies directed against nitrosylated epitopes have been found in sera from patients suffering from human African trypanosomiasis (HAT) but not in sera from control subjects living in the same endemic area or African control subjects living in France. We conjugated amino acids to albumin by glutaraldehyde (conjugates) and then nitrosylated the conjugates. Both conjugates and nitrosylated conjugates were analysed by enzyme-linked immunosorbent assay (ELISA). We detected antibodies directed against nitrosylated L-cysteine and L-tyrosine conjugates; antibody levels were higher in stage II patients than in stage I. Patients with severe clinical signs had higher antibody levels, and antibody levels were highest in patients with major neurological signs. Antibody response was only associated with the IgM isotype. We evaluated antibody specificity and avidity by competition experiments using conjugates and nitrosylated conjugates. Avidity was around 2 x10(-6) m for the S-nitroso-cysteine epitope and 2 x 10(-8) m for the S-nitroso-tyrosine epitope. Detection of circulating antibodies to S-nitroso-cysteine and S-nitroso-tyrosine epitopes provides indirect evidence for nitric oxide (NO) involvement in HAT and their levels are correlated with disease severity.

Experimental studies on the evolution of antimony-resistant phenotype during the in vitro life cycle of Leishmania infantum: implications for the spread of chemoresistance in endemic areas.

Pentavalent antimonial unresponsiveness is an emerging problem in endemic areas and information on factors which could modulate the transmission of drug-resistant phenotypes and parasites during life cycle are warranted. Using axenic amastigotes resistant to potassium antimonyl tartrate (Sb(III)) we investigated the modulation of antimonyl resistance during the in vitro life cycle. We assessed: (i) the stability of the drug-resistant phenotype during the in vitro life cycle; (ii) the transmission of drug-resistant clones when mixed with a wild-type clone at different susceptible/chemoresistant ratios (50/50,90/10,10/90) after one or two in vitro life cycles. We demonstrate that: (i) mutants which were 12,28,35 and 44 fold more resistant to Sb(III)-antimonial than their parental wild-type, were Glucantime Sb(V)-resistant when growing in THP-1 cells; (ii) the drug-resistant phenotype was partially retained during long-term in vitro culture (3 months) in drug free medium; (iii) the antimonyl-resistant phenotype was retained after one or more in vitro life cycles. However, when drug-resistant parasites were mixed with susceptible, mutants could not be detected in the resulting population, after one or two in vitro life cycles, whatever the initial wild-type/chemoresistant ratio. These results could be explained by the lower capacity of drug-resistant amastigotes to undergo the amastigote-promastigote differentiation process, leading probably to their sequential elimination during life cycle. Taken together, these observations demonstrate that different factors could modulate the transmission of Leishmania drug resistance during the parasite's life cycle.

Antimonial-mediated DNA fragmentation in Leishmania infantum amastigotes.

The basic treatment of leishmaniasis consists in the administration of pentavalent antimonials. The mechanisms that contribute to pentavalent antimonial toxicity against the intracellular stage of the parasite (i.e., amastigote) are still unknown. In this study, the combined use of several techniques including DNA fragmentation assay and in situ and cytofluorometry terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling methods and YOPRO-1 staining allowed us to demonstrate that potassium antimonyl tartrate, an Sb(III)-containing drug, was able to induce cell death associated with DNA fragmentation in axenic amastigotes of Leishmania infantum at low concentrations (10 microg/ml). This observation was in close correlation with the toxicity of Sb(III) species against axenic amastigotes (50% inhibitory concentration of 4.75 microg/ml). Despite some similarities to apoptosis, nuclease activation was not a consequence of caspase-1, caspase-3, calpain, cysteine protease, or proteasome activation. Altogether, our results demonstrate that the antileishmanial toxicity of Sb(III) antimonials is associated with parasite oligonucleosomal DNA fragmentation, indicative of the occurrence of late events in the overall process of apoptosis. The elucidation of the biochemical pathways leading to cell death could allow the isolation of new therapeutic targets.

In vitro growth of Leishmania amazonensis promastigotes resistant to pentamidine is dependent on interactions among strains.

The in vitro growth of promastigote cells of Leishmania amazonensis was found to strongly depend on interactions among strains that differed in their pentamidine resistance. In particular, the growth of resistant strains was reduced when they shared the same environment with a less-resistant strain.

Efficacy of second line drugs on antimonyl-resistant amastigotes of Leishmania infantum.

In a previous paper we have demonstrated that the induction, by direct drug pressure, of a resistance to Sb(III) antimony at physiological concentration in the amastigote stage of the parasite, led to a high cross-resistance to Sb(V) species in the form of Glucantime. In this paper, further chemoresistant clones were characterized. Axenic amastigotes of Leishmania infantum were adapted to survive in culture medium containing 4, 20, 30 and 120 microg/ml of potassium antimonyl tartrate Sb(II). These mutants were 12, 28, 35 and 44-fold more resistant to Sb(III) than the parental wild-type clone. They were able to resist at concentrations of Glucantime Sb(V) as high as 160 microg/ml when growing in THP-1 cells. We have investigated the efficacy of second line drugs in clinical use (pentamidine and amphotericin B) on the antimony-resistant mutants. Amphotericin B was toxic for both wild-type and chemoresistant mutants at concentrations ranging from 0.05 to 0.15 microM. Pentamidine which is extensively used when the first course of antimonial pentavalent compounds is unsuccessful, was more toxic for all the chemoresistant organisms than for the wild-type clone. In the same way, chemoresistant amastigotes growing within THP-1 cells were more susceptible to pentamidine than the wild-type clone. Our results showed that the resistance of the mutants was restricted to the antimony containing drugs and did not led to a cross-resistance against the other clinically relevant drugs. These results confirmed that these two drugs (pentamidine and amphotericin B) are good candidates to treat pentavalent antimonial unresponsiveness.

Temperature- and pH-dependence of proton relaxation rates in rat liver tissue.

In this study proton NMR relaxation rates R1 and R2 and pH of untreated fresh rat liver tissue as well as changes up to 4 h after tissue excision was investigated. Measurements were performed in the temperature range from 7 to 45 degrees C. Dynamic changes in all parameters were observed at higher temperatures (> or = 30 degrees C). A qualitatively different time course after tissue excision of all three parameters was obtained for 45 degrees C. The relative alterations like the change of relaxation rates referred to a reference value can predict tissue viability. A good correlation between R2 and pH in the range of 7-37 degrees C was detected and is described by an improved analytical model for R2 as a function of temperature and pH. The observed data lead to an empirical model given by R2 (pH,T) = 39.8 + (10.2 * delta pH) + (0.1 * delta T) + (0.2 * delta pH delta T), where the reference pH value was chosen 7.4 and the reference temperature value 37 degrees C (310.15 K), respectively.

Proton spin-spin relaxation times as liver transplantation graft viability parameter.

In this study, spin-spin relaxation time T2 of stored rat liver grafts was investigated and correlated with other viability parameters. Five different storage solutions were applied and T2 as well as overall tissue water content, bile flow, and energy charge were determined for increasing durations of cold storage (1-48 hr). Good correlation between T2 and tissue water content was detected. In a subset of experiments, dealing with additional warm ischemia after cold storage, the relative increase of T2 and energy charge showed a very good correlation. The results suggest the possible use of relaxation time parameters as liver graft viability parameters in a combined protocol of rapid viability assays for human liver transplantation surgery.

Proton spin-lattice relaxation time as liver transplantation graft viability parameter.

In this study proton NMR T1 of stored rat liver grafts was investigated and correlated with other viability parameters. Five different storage solutions were used and T1 as well as overall tissue water content, bile flow, and energy charge were determined after various periods of cold storage (1 to 48 hr). A good correlation between T1 and tissue water content was detected, suggesting the possible use of this relaxation time parameter as liver graft viability parameter in a combined protocol of rapid viability assays for human liver transplantation surgery.

Comparison of HTK- and UW-solution for liver preservation tested in an orthotopic liver transplantation model in the pig.

The aim of this experimental study was to compare the preservation potency of University of Wisconsin (UW) and HTK (Bretschneider) solutions in an orthotopic liver transplantation (OLT) model in pigs. Livers were harvested using an in situ perfusion technique, where organs were flushed with the solution being tested, stored on ice--cold storage (CS)--for 2 or 24 h and then transplanted. Parameters monitored were liver enzymes in serum, hepatic water content, high energy phosphates, nuclear magnetic resonance (NMR) relaxation time T2, light microscopy and bile production. CS for 24 h is an extreme in pig liver preservation and is not compatible with animal survival. Biopsies showed drastic morphological changes and grafts did not produce bile in either group. (Bile production 2 h CS: HTK, 5.6 +/- 1.8 ml/h; UW, 4.7 +/- 2.3 ml/h) Enzyme release after reperfusion (deltaSGOT, deltaLDH) was higher in long-term preservation. Hepatic tissue water content significantly decreased during CS in UW preserved livers. Edema alter reperfusion (deltaH2O: HTK 24 h = +5.6%, UW 24 h = +4.8%) and regeneration capacity after reperfusion (UW 2 h = 63%, HTK 2 h = 55%, UW 24 h = 30%, HTK 24 h = 30%) were not significantly different. However, we did not observe major differences in preservation potency between the solutions tested. Differences were correlated, rather, with length 9 time of CS, than with the solution used. Therefore, HTK solution seemed to be a low potassium containing alternative to UW solution.

In vitro NMR investigation of controlled single and repeated isoflurane anesthesia.

Immobilization of laboratory animals is a basic requirement for experimental in vivo NMR measurements. The effect of single and repeated isoflurane anesthesia on proton NMR relaxation times T1 and T2 in rat liver was studied. Furthermore, physiological monitoring was performed to evaluate the influence of isoflurane anesthesia (up to 2 hr) on biological parameters. Neither single nor repeated isoflurane application over the observed time produce relevant alterations of physiological parameters or relaxation times, compared with untreated control groups. Therefore, we conclude that isoflurane anesthesia is appropriate for in vivo NMR investigations, especially of the liver.