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D Le Ray

Publications and source records attributed to D Le Ray.

At least 19 recordsLinked to original sources
PubMed2006

Clonal propagation and the fast generation of karyotype diversity: An in vitro Leishmania model.

In the present work we studied the karyotype stability during long-term in vitro maintenance in 3 cloned strains of Leishmania (Viannia) peruviana, Leishmania (Viannia) braziliensis and a hybrid between both species. Only the L. (V.) peruviana strain showed an unstable karyotype, even after subcloning. Four chromosomes were studied in detail, each of them characterized by homologous chromosomes of different size (heteromorphy). Variations in chromosome patterns during in vitro maintenance were rapid and discrete, involving loss of heteromorphy or appearance of additional chromosome size variants. The resulting pattern was not the same according to experimental conditions (subinoculation rate or incubation temperature), and interestingly, this was associated with differences in growth behaviour of the respective parasites. No change in total ploidy of the cells was observed by flow cytometry. We discuss several mechanisms that might account for this variation of chromosome patterns, but we favour the occurrence of aneuploidy, caused by aberrant chromosome segregation during mitosis. Our results provide insight into the generation of karyotype diversity in natural conditions and highlight the relativity of the clone concept in parasitology.

Animals↗
PubMed2005

American tegumentary leishmaniasis: antigen-gene polymorphism, taxonomy and clinical pleomorphism.

Multi-locus enzyme electrophoresis is the current gold standard for the genetic characterisation of Leishmania. However, this method is time-consuming and, more importantly, cannot be directly applied to parasites present in host tissue. PCR-based methods represent an ideal alternative but, to date, a multi-locus analysis has not been applied to the same sample. This has now been achieved with a sample of 55 neotropical isolates (Leishmania (Viannia) braziliensis, L. (V.) peruviana, L. (V.) guyanensis, L. (V.) lainsoni and L. (L.) amazonensis), using five different genes as targets, four of which encoded major Leishmania antigens (gp63, Hsp70, H2B and Cpb). Our multi-locus approach strongly supports the current taxonomy and demonstrates a highly robust method of distinguishing different strains. Within L. (V.) braziliensis, we did not encounter so far specific genetic differences between parasites isolated from cutaneous and mucosal lesions. Interestingly, results provided by each of the different antigen-genes in the species considered, were different, suggesting different selective pressures. Our work emphasises the need for a multi-disciplinary approach to study the clinical pleomorphism of leishmaniasis.

Animals↗
PubMed2005

Complexity of the major surface protease (msp) gene organization in Leishmania (Viannia) braziliensis: evolutionary and functional implications.

The major surface protease (msp or gp63) of Leishmania plays a major role in the host-parasite interaction. We analysed here the structure of the msp gene locus in Leishmania (Viannia) braziliensis and compared it to results obtained in other species. Physical mapping of cosmid contigs revealed a minimum of 37 genes per haploid genome and at least 8 different msp gene families. Within the same organism, these genes showed a nucleotide sequence varying in certain stretches from 3 to 34%, and a mosaic structure. From an evolutionary point of view, major differences were observed between subgenera Viannia and Leishmania, both in terms of msp gene number and sequence. Within subgenus Viannia, phenetic analysis revealed three clusters in which sequence variants of L. (Viannia) braziliensis and L. (Viannia) guyanensis were interspersed. Functional implications of our results were explored from predicted L. (Viannia) braziliensis protein sequences: regions encoding the msp catalytic site showed a conserved sequence, while regions encoding surface domains possibly involved in the host-parasite interaction (macrophage adhesion sites and immunodominant B-cell and T-cell epitopes) were variable. We speculate that this would be an adaptive strategy of the parasite.

Animals↗
PubMed2004

Evaluation of a urinary antigen-based latex agglutination test in the diagnosis of kala-azar in eastern Nepal.

BACKGROUND: We evaluated the diagnostic accuracy as well as the reproducibility of the urine latex agglutination test 'KAtex' in the diagnosis of kala-azar in patients recruited at a tertiary care centre in Dharan, Nepal, between November 2000 and January 2002. METHODS: All patients presenting with fever of 2 weeks or more and splenomegaly were consecutively enrolled. Bone marrow and--if negative--spleen aspirates were examined for Leishmania donovani. Serum and urine samples were taken in duplicate for the Direct Agglutination Test (DAT) and KAtex. The reference laboratory determined sensitivity and specificity of KAtex. Reproducibility between both laboratories was assessed. RESULTS: KAtex was performed on urine from 155 parasitologically confirmed kala-azar and 77 non-kala-azar cases (parasitology and DAT-negative). KAtex showed a sensitivity of 47.7% (74/155, 95% CI: 39.7-55.9) and a specificity of 98.7% (76/77, 95% CI: 93.0-100.0). Reproducibility of KAtex showed a kappa of 0.684 (P < 0.001, n = 232). CONCLUSION: KAtex evaluation showed high specificity, low sensitivity and moderate reproducibility. A urine test for kala-azar could become a real breakthrough in kala-azar management if its reproducibility and sensitivity could be further improved.

Adult↗
PubMed2003

Prospective evaluation and comparison of the direct agglutination test and an rK39-antigen-based dipstick test for the diagnosis of suspected kala-azar in Nepal.

The diagnosis of visceral leishmaniasis (kala-azar) remains difficult in rural endemic areas and practical and reliable tests are badly needed. Two serological tests, the Direct Agglutination Test (DAT) and an rK39-antigen-based dipstick test, were compared to parasitological diagnosis in a group of 184 patients presenting at a tertiary care centre in south-eastern Nepal with a history of fever > or = 14 days and splenomegaly; 139 patients had a parasitologically proven kala-azar and 45 patients had a negative parasitological work-up. The rK39 dipstick showed a sensitivity of 97% and a specificity of 71%. The DAT was up to 99% sensitive with a low cut-off titre (1:400) but its specificity did not exceed 82% even with a high cut-off titre (1:51 200). Both tests could be used for screening suspect patients in endemic areas. However, their use as confirmatory tests should be restricted to situations where the proportion of kala-azar among clinical suspect patients is high. The rK39 dipstick is cheaper and easier to use than the DAT and could be used widely provided that both its performance and production remain stable.

Adult↗
PubMed2002

Molecular epidemiology and diagnosis of Leishmania: what have we learnt from genome structure, dynamics and function?

This paper reviews our exploration of the dynamics of the Leishmania genome and its contribution to epidemiology and diagnosis. We used as a model Peruvian populations of L. (Viannia) braziliensis and L. (V.) peruviana, 2 species very close phylogenetically, but phenotypically very different in biotope and pathology. We initially focused on karyotype analysis. Our data showed that chromosomes were subject to a fast rate of evolution, and were sensitive indicators of genetic drift. Therefore, molecular karyotyping appeared an adequate tool for monitoring (i) emergence of close species, (ii) ecogeographical differentiation at the intraspecific level, and (iii) strain 'fingerprinting'. Chromosome size variation was mostly due to the number of tandemly repeated genes (rDNA, mini-exon, gp63, and cysteine proteinase genes), and could involve the deletion of unique genes (L. (V.) braziliensis-specific gp63 families). Considering the importance of these genes in parasitism, their rearrangement might have functional implications: adaptation to different environments and pleomorphic pathogenicity. Our knowledge of genome structure and dynamics was used to develop new polymerase chain reaction (PCR) techniques. Amplification of gp63 genes followed by cleavage with restriction enzymes and study of restriction fragment length polymorphism (gp63 PCR-RFLP) allowed the discrimination of all species tested, even directly in biopsies with 95% sensitivity (compared with PCR amplification of kinetoplast deoxyribonucleic acid). At the intra-specific level, RFLP was also observed and corresponded to mutations in major immunogen domains of gp63. These seem to be under strong selection pressure, and the technique should facilitate addressing how the host's immune pressure may modulate parasite population structure. Altogether, gp63 PCR-RFLP represents a significant operational improvement over the other techniques for molecular epidemiology and diagnosis: it combines sensitivity, discriminatory power and prognostic value.

Animals↗
PubMed2002

How better drugs could change kala-azar control. Lessons from a cost-effectiveness analysis.

Conditional on correct diagnosis and treatment, current drug regimens for visceral leishmaniasis (VL) will only prevent about 90% of deaths. Furthermore, the cost of pentavalent antimonials, the long duration of the regimen and its parenteral administration are major obstacles for patients. Poor patient compliance and the use of counterfeit drugs contribute to therapeutic failure, amplification of the reservoir and the appearance of drug resistance. We assessed the impact of potential improvements in chemotherapy on the cost-effectiveness of VL test-treatment strategies. Competing test-treatment strategies were compared in a formal decision analysis - from the viewpoint of the clinician facing a VL suspect -, with avoided VL-mortality and cost as outcomes of interest. Sensitivity analysis was done involving the following parameters: efficacy, toxicity and cost of treatment including patient care. When safer and more efficacious drugs are considered, they only result in a more cost-effective strategy if the total cost of treatment falls below US$ 390 per patient. A serological test-treatment strategy remains the optimal choice, also when better drugs become available.

Agglutination Tests↗
PubMed2001

Adaptive motor control in crayfish.

This article reviews the principles that rule the organization of motor commands that have been described over the past five decades in crayfish. The adaptation of motor behaviors requires the integration of sensory cues into the motor command. The respective roles of central neural networks and sensory feedback are presented in the order of increasing complexity. The simplest circuits described are those involved in the control of a single joint during posture (negative feedback-resistance reflex) and movement (modulation of sensory feedback and reversal of the reflex into an assistance reflex). More complex integration is required to solve problems of coordination of joint movements in a pluri-segmental appendage, and coordination of different limbs and different motor systems. In addition, beyond the question of mechanical fitting, the motor command must be appropriate to the behavioral context. Therefore, sensory information is used also to select adequate motor programs. A last aspect of adaptability concerns the possibility of neural networks to change their properties either temporarily (such on-line modulation exerted, for example, by presynaptic mechanisms) or more permanently (such as plastic changes that modify the synaptic efficacy). Finally, the question of how "automatic" local component networks are controlled by descending pathways, in order to achieve behaviors, is discussed.

Adaptation, Physiological↗
PubMed2001

Genomic polymorphism of Leishmania infantum: a relationship with clinical pleomorphism?

Leishmania infantum is the etiological agent of visceral (VL) and a cutaneous form (CL) of leishmaniasis around the Mediterranean Basin. In order to document the parasite genetic background corresponding to this clinical diversity, chromosome size polymorphism was analysed in 32 French isolates (18 CL and 14 VL) originating from the Cévennes and the Pyrénées Orientales (PO), and corresponding to zymodemes MON-1 and MON-29. Five chromosomes bearing tandemly repeated genes encoding for important antigens (gp63, PSA-2 and K39) or key metabolic functions (mini-exon and rDNA) were studied. Significant size variation (100-270 kbp) was observed for chromosomes bearing mini-exon, PSA-2 and rDNA genes, which involved variation in copy number of corresponding genes. The two other chromosomes showed smaller size-variation and did not involve dosage of gp63 and K39 genes. Chromosomal size showed correlation with geography and clinical origin: (i) chromosome 2 (mini-exon) was found to be significantly smaller in the PO; (ii) chromosomes 12 (PSA-2) and 27 (rDNA) were significantly smaller in the strictly cutaneous MON-29 isolates. Gene rearrangements and their synergistic effects on the phenotypic expression of the parasite are discussed.

Animals↗
PubMed2001

Gp63 gene polymorphism and population structure of Leishmania donovani complex: influence of the host selection pressure?

The gp63 encoding genes were characterized by PCR-RFLP in 35 isolates representative of the Leishmania donovani complex (L. infantum, L. donovani, L. archibaldi and L. chagasi), with special attention to Mediterranean L. infantum from different geographical origins, and in separate groups from Old World Leishmania (L. major, L. tropica and L. aethiopica). The aim was to evaluate how the possible selective pressure by the host on these important surface proteins would influence structuring of our sample. Comparison was carried out with the structure obtained (i) from reported isoenzyme data, characters supposed to vary neutrally, and (ii) from PCR-RFLP analysis of gp63 inter-genic regions, containing nontranslated spacers and regulatory genes. Polymorphism within the gp63-encoding region, was much higher than in gp63 inter-genic regions. In the gp63 intra-genic dendrogram, the 4 species of L. donovani complex were discriminated and quite distinct from outgroups. Within L. infantum, geographical structuring was observed and did not overlap with the structure built-up from isoenzymes and inter-genic data. These results support the idea of a strong host-selection on gp63, at vector level but most of all at vertebrate (human or dog) immunological level. Furthermore, they illustrate how the nature of genetic characters may influence the perception of population structuring.

Algeria↗
PubMed2000

A cellular mechanism for the transformation of a sensory input into a motor command.

The initiation and control of locomotion largely depend on processing of sensory inputs. The cellular bases of locomotion have been extensively studied in lampreys where reticulospinal (RS) neurons constitute the main descending system activating and controlling the spinal locomotor networks. Ca(2+) imaging and intracellular recordings were used to study the pattern of activation of RS neurons in response to cutaneous stimulation. Pressure applied to the skin evoked a linear input/output relationship in RS neurons until a threshold level, at which a depolarizing plateau was induced, the occurrence of which was associated with the onset of swimming activity in a semi-intact preparation. The occurrence of a depolarizing plateau was abolished by blocking the NMDA receptors that are located on RS cells. Moreover, the depolarizing plateaus were accompanied by a rise in [Ca(2+)](i), and an intracellular injection of the Ca(2+) chelator BAPTA into single RS cells abolished the plateaus, suggesting that the latter are Ca(2+) dependent and rely on intrinsic properties of RS cells. The plateaus were shown to result from the activation of a Ca(2+)-activated nonselective cation current that maintains the cell in a depolarized state. It is concluded that this intrinsic property of the RS neuron is then responsible for the transformation of an incoming sensory signal into a motor command that is then forwarded to the spinal locomotor networks.

Action Potentials↗
PubMed2000

Voltage-clamp analysis of the potentiation of the slow Ca2+-activated K+ current in hippocampal pyramidal neurons.

Exploring the principles that govern activity-dependent changes in excitability is an essential step to understand the function of the nervous system, because they act as a general postsynaptic control mechanism that modulates the flow of synaptic signals. We show an activity-dependent potentiation of the slow Ca2+-activated K+ current (sl(AHP)) which induces sustained decreases in the excitability in CA1 pyramidal neurons. We analyzed the sl(AHP) using the slice technique and voltage-clamp recordings with sharp or patch-electrodes. Using sharp electrodes-repeated activation with depolarizing pulses evoked a prolonged (8-min) potentiation of the amplitude (171%) and duration (208%) of the sl(AHP). Using patch electrodes, early after entering the whole-cell configuration (<20 min), responses were as those reported above. However, although the sl(AHP) remained unchanged, its potentiation was markedly reduced in later recordings, suggesting that the underlying mechanisms were rapidly eliminated by intracellular dialysis. Inhibition of L-type Ca2+ current by nifedipine (20 microM) markedly reduced the sl(AHP) (79%) and its potentiation (55%). Ryanodine (20 microM) that blocks the release of intracellular Ca2+ also reduced sl(AHP) (29%) and its potentiation (25%). The potentiation of the sl(AHP) induced a marked and prolonged (>50%; approximately equals 8 min) decrease in excitability. The results suggest that sl(AHP) is potentiated as a result of an increased intracellular Ca2+ concentration ([Ca2+]i) following activation of voltage-gated L-type Ca2+ channels, aided by the subsequent release of Ca2+ from intracellular stores. Another possibility is that repeated activation increases the Ca2+-binding capacity of the channels mediating the sl(AHP). This potentiation of the sl(AHP) could be relevant in hippocampal physiology, because the changes in excitability it causes may regulate the induction threshold of the long-term potentiation of synaptic efficacy. Moreover, the potentiation would act as a protective mechanism by reducing excitability and preventing the accumulation of intracellular Ca2+ to toxic levels when intense synaptic activation occurs.

Action Potentials↗
PubMed2000

Central control components of a 'simple' stretch reflex.

The monosynaptic stretch reflex is a fundamental feature of sensory-motor organization in most animal groups. In isolation, it serves largely as a negative feedback devoted to postural controls; however, when it is involved in diverse movements, it can be modified by central command circuits. In order to understand the implications of such modifications, a model system has been chosen that has been studied at many different levels: the crayfish walking system. Recent studies have revealed several levels of control and modulation (for example, at the levels of the sensory afferent and the output synapse from the sensory afferent, and via changes in the membrane properties of the postsynaptic neuron) that operate complex and highly adaptive sensory-motor processing. During a given motor task, such mechanisms reshape the sensory message completely, such that the stretch reflex becomes a part of the central motor command.

Animals↗
PubMed1999

Active motor neurons potentiate their own sensory inputs via glutamate-induced long-term potentiation.

Adaptive motor control is based mainly on the processing and integration of proprioceptive feedback information. In crayfish walking leg, many of these operations are performed directly by the motor neurons (MNs), which are connected monosynaptically by sensory afferents (CBTs) originating from a chordotonal organ that encodes vertical limb movements. An in vitro preparation of the crayfish CNS was used to investigate a new control mechanism exerted directly by motor neurons on the sensory inputs themselves. Paired intracellular recordings demonstrated that, in the absence of any presynaptic sensory firing, the spiking activity of a leg MN is able long-lastingly to enhance the efficacy of the CBT-MN synapses. Moreover, this effect is specific to the activated MN because no changes were induced at the afferent synapses of a neighboring silent MN. We report evidence that long-term potentiation (LTP) of the monosynaptic EPSP involves a retrograde system of glutamate transmission from the postsynaptic MN, which induces the activation of a metabotropic glutamate receptor located presynaptically on the CBTs. We demonstrate that LTP at crayfish sensory-motor synapses results exclusively from the long-lasting enhancement of release of acetylcholine from presynaptic sensory afferent terminals, without inducing any modifications in postsynaptic MN properties. Our data indicate that this positive feedback control represents a functional mechanism that may play a key role in the auto-organization of sensory-motor networks.

Animals↗
PubMed1999

In vitro promastigote fitness of putative Leishmania (Viannia) braziliensis/Leishmania (Viannia) peruviana hybrids.

In order to initiate studies on the phenotypic properties of hybrids vs. their putative parents, the in vitro growth behaviour of promastigotes was compared for 15 stocks characterised as Leishmania (Viannia) braziliensis, Leishmania (Viannia) peruviana and putative hybrids (isolated from the Eastern Andean valley of Huanuco, Peru). Five sets of three stocks, each set including a L.(V.)braziliensis, a L.(V.)peruviana and a putative hybrid, were constituted randomly and counted daily close to isolation from man (ten to 18 subcultures). Hybrids and L.(V.)peruviana presented similar growth characteristics, and they displayed a growth capacity (growth rate and cell density at stationary phase) significantly lower than the one of L.(V.)braziliensis. Following prolonged in vitro maintenance of one of the sets, the hybrid kept its lower growth capacity. The contrast between the difficulty to grow in vitro these putative hybrids, and their high isolation rate from natural populations is discussed.

Animals↗
PubMed1999

Size-polymorphism of mini-exon gene-bearing chromosomes among natural populations of Leishmania, subgenus Viannia.

In order to explore genomic plasticity at the level of the mini-exon gene-bearing chromosome in natural populations of Leishmania, the molecular karyotype of 84 Leishmania stocks belonging to subgenus Viannia, originating mostly from Peru and Bolivia, and differing according to eco-geographical and clinical parameters, was resolved and hybridised with a mini-exon probe. The results suggest that size variation of the mini-exon gene-bearing chromosome is frequent and important (up to 245-kb size-difference), and partially involves variation (up to 50%) in copy number of mini-exon genes. There is no significant size-difference between mini-exon-bearing chromosomes of Peruvian and Bolivian populations of cutaneous and mucosal isolates of Leishmania (Viannia) braziliensis, but there is between eco-geographical populations of Leishmania (Viannia) peruviana. Leishmania (V.) peruviana presented a significantly smaller mini-exon-bearing chromosome than the other species of subgenus Viannia. The contrast between the general chromosome size heterogeneity and the homogeneity observed in some Peruvian Andean areas is discussed in terms of selective pressure.

Animals↗