DEAD-box proteins, like Leishmania eIF4A, modulate interleukin (IL)-12, IL-10 and tumour necrosis factor-alpha production by human monocytes.

Previously we showed that His-tagged, recombinant, Leishmania infantum eukaryotic initiation factor (LeIF) was both an RNA-dependent ATPase and an ATP-dependent RNA helicase in vitro, as described for other members of the DEAD-box helicase family. In addition, we showed that LeIF induces the production of IL-12, IL-10, and TNF-α by human monocytes. This study aims to characterize the cytokine-inducing activity in human monocytes of several proteins belonging to the DEAD-box family from mammals and yeast. All tested proteins contained the 11 conserved motifs (Q, I, Ia, GG Ib, II, III, IV, QxxR, V and VI) characteristic of DEAD-box proteins, but they have different biological functions and different percentages of identities with LeIF. We show that these mammalian or yeast recombinant proteins also are able to induce IL-12, IL-10 and TNF-α secretion by monocytes of healthy human subjects. This cytokine-inducing activity is proteinase K sensitive and polymyxin B resistant. Our results show that the induction of cytokines in human monocytes is not unique to the protein LeIF of Leishmania, and it suggests that the activity of certain DEAD-box proteins can be exploited as adjuvant and/or to direct immune responses towards a Th1 profile in vaccination or immunotherapy protocols.

In vitro growth kinetics, differentiation and morphological characterisation of Tunisian Leishmania infantum parasites.

In this study, a negative peanut agglutinin (PNA) selection was used as a marker for promastigote differentiation to compare the in vitro growth and differentiation kinetics of two visceral and two cutaneous Leishmania (Leishmania) infantum parasites. All parasites had different growth and differentiation kinetics. Cultures initiated with PNA(+) parasites purified during the early stationary phase (Day 4), when PNA(-) (non-agglutinating) parasites peaked, yielded a high PNA(-) percent. Further morphological analysis at this time point showed that 60-86% of PNA(+) forms were procyclics, whilst PNA(-) forms were composed of 53-71% leptomonads. Nectomonads were present both in PNA(-) and PNA(+) promastigote fractions at nearly equivalent proportions, suggesting that they constitute a transition state in the Leishmania development process, with a fraction of them sharing common constituents of the surface coat with procyclics and the other with leptomonads. Obtaining a high density of promastigotes undergoing developmental differentiation may be useful for further molecular and biochemical identification of developmental stage-specific markers.

Evaluation of antileishmanial, cytotoxic and antioxidant activities of essential oils extracted from plants issued from the leishmaniasis-endemic region of Sned (Tunisia).

In this study, we tested 10 essential oils (EOs) extracted from 10 plants issued from Sned region (Tunisia) to evaluate both their leishmanicidal effects against Leishmania major and L. infantum, and their cytotoxicity against murine macrophage cell line RAW 264.7 (ATCC, TIB-71). The antioxidant activity was also monitored by the DDPH method, while the chemical composition of active EO was assessed by GC-MS analysis. The results showed that the EOs obtained from Thymus hirtus sp. algeriensis (rich on monoterpenoids, especially linalool at 17.62% and camphor at 13.82%) is significantly active against both L. major and L. infantum, whereas Ruta chalepensis EO (rich on 2-undecanone at 84.28%) is only active against L. infantum. Both oil extracts showed low cytotoxicity towards murine macrophages. The characteristic ratios (IC80 Raw264.7 cells/IC50 L. infantum and IC80 Raw264.7 cells/IC50 L. major) were, respectively, 2.7 and 1.57 for T. hirtus sp. algeriensis, and 1.34 and 0.19 for R. chalepensis. However, when measuring the antioxidant effects (DDPH method), the two latter EOs presented a moderate 2,2-diphenyl-2-picrylhydrazyl hydrate scavenging effects compared to EOs from Eucaliptus globulus, Pinus halepensis, Pituranthos tortuosus, Rosmarinus officinalis, Tetraclinis articulata or to BHT.

Leishmania infantum LeIF and its recombinant polypeptides modulate interleukin IL-12p70, IL-10 and tumour necrosis factor-α production by human monocytes.

Leishmania eukaryotic initiation factor (LeIF) antigen, a Leishmania protein, was shown to induce IL-12, IL-10 and tumour necrosis factor-α (TNF-α) production by human monocytes-derived macrophages and dendritic cells from healthy individuals. This cytokine-inducing activity was previously found to be located in the amino-terminal region of LeIF protein. This study aimed at characterizing the cytokine-inducing activity of Leishmania infantum LeIF [Leishmania (L.) infantum (LieIF)] and at defining the fragments necessary for inducing cytokine secretion. Eleven rationally designed recombinant polypeptides, corresponding to the entire LeIF protein or parts of it, were expressed and used to stimulate monocytes from healthy individuals. Leishmania (L.) infantum was able to induce IL-12p70, IL-10 and TNF-α secretion in human monocytes. In addition, both amino- (1-226) and carboxyl-terminal (196-403) parts of the protein were shown to induce significant levels of the three cytokines analysed. However, IL-12p70-inducing activity was not significant when monocytes were stimulated with the fragments 129-226 and 129-261, inferring that IL-12p70-inducing activity was primarily located within amino acids 1-129 and 261-403. Although the full-length LieIF protein was a more potent inducer than the tested fragments, a significant cytokine-inducing activity was maintained in smaller amino acid regions. This work suggests that cytokine-inducing activity of LieIF or its parts could be exploited in vaccination or immunotherapy protocols.

Differences in the salivary effects of wild-caught versus colonized Phlebotomus papatasi (Diptera: Psychodidae) on the development of zoonotic cutaneous leishmaniasis in BALB/c mice.

Preimmunization of mice with salivary gland homogenate (SGH) of long-term colonized (F29) female Phlebotomus papatasi Scopoli (Diptera: Psychodidae) induced protection against Leishmania major Yakimoff & Schokhor (Kinetoplastida: Trypanosomatidae) co-inoculated with the same type of SGH. In contrast, preimmunization of mice with SGH of wild-caught female P. papatasi did not confer protection against L. major co-inoculated with the same type of SGH. Similarly, SGH from recently colonized (F1) female P. papatasi did not protect mice against L. major. These results suggest that when developing a sand fly saliva-based vaccine, the natural vector populations should be considered.

Spatial correlation between Phlebotomus papatasi Scopoli (Diptera: Psychodidae) and incidence of zoonotic cutaneous leishmaniasis in Tunisia.

The geographical distribution of Phlebotomus papatasi Scopoli, vector of Leishmania major Yakimoff and Schokhor (Kinetoplastida: Trypanosomatidae), the etiologic agent of zoonotic cutaneous leishmaniasis (ZCL), was assessed during September 2006 through a transect from the north to the south of Tunisia using CDC light traps. P. papatasi was found to be abundant in the arid and Saharan bioclimatic zones and rare in the humid, subhumid, and semiarid bioclimatic zones. Similarly, the highest incidence of ZCL was observed in the arid and Saharan bioclimatic zones and the lowest in the humid, subhumid, and semiarid bioclimatic zones. Our overall findings confirm the close spatial association between the abundance of P. papatasi and the incidence of ZCL.

Comparative evaluation of specific ELISA and RFFIT antibody assays in the assessment of dog immunity against rabies.

Two techniques are currently used to evaluate the humoral immune responses to rabies vaccination: ELISA, which detects binding antibodies to viral antigens and the WHO reference rapid fluorescent focus inhibition test (RFFIT), which assays in vitro virus-neutralizing antibodies. In this study, we have comparatively evaluated antibody responses of dogs reared either in an experimental kennel or living in field conditions after vaccination with a cell culture-derived rabies vaccine. In experimental conditions, both ELISA and RFFIT techniques were well correlated. However, in field conditions, they yielded discrepant results particularly in evaluating the residual rabies immunity before vaccine administration and in identifying seroconverted dogs. After rabies vaccination in field conditions, while similar antibody titres and seroconversion rates were obtained using either technique, the discrimination of a given dog according to the seroconversion threshold depended on the assay. We concluded, that whereas in experimental conditions, ELISA and RFFIT were well correlated, in field conditions ELISA yielded upper estimates. Consequently, RFFIT, although a cumbersome test, should continue to be considered as the reference rabies antibody assay technique. A seroconversion threshold of 0.5 IU/ml should be cautiously considered and a higher threshold (1 IU/ml) could be more appropriate in the evaluation of rabies immunity in the field in order to marginalize the interfering factors.

[Association study between diabetic retinopathy and aldose reductase gene polymorphism in Tunisians]. / Etude de l'association entre la rétinopathie diabétique et un polymorphisme du gène de l'aldose réductase dans la population tunisienne.

INTRODUCTION: Aldose reductase (ALR2), the enzyme of the polyol pathway, may play an important role in the pathogenesis of diabetic microvascular complications, namely diabetic retinopathy. The study aimed to determine whether the aldose reductase gene is involved in diabetic retinopathy in the Tunisian population. MATERIAL: and methods: A case-control study was conducted in 47 type 2 diabetic patients who have diabetic retinopathy and 28 diabetic patients without diabetic retinopathy in spite of diabetes lasting for more than 5 years and over 10 years in 13 cases. We investigated the association between the (CA)n polymorphism located at 2.1 kb upstream of the transcription start site of ALR2 and diabetic retinopathy. The distribution of genotypes and alleles was compared between cases and controls by chi2 test using Epi info software. RESULTS: Genotyping of the two groups did not demonstrate any association between the alleles of this marker and diabetic retinopathy in the Tunisian population studied. DISCUSSION: An association between one of the alleles (Z - 2) of this microsatellite and diabetic retinopathy was identified in Chinese and Japanese patients with type 2 diabetes. Discordant results were obtained for the different populations studied. The lack of an association between diabetic retinopathy and ALR2 alleles indicates that the ALR2 gene is not a genetic marker of predisposition to diabetic retinopathy for type 2 diabetic patients in the Tunisian population studied.

Association analysis of HLA-class II and class III gene polymorphisms in the susceptibility to mediterranean visceral leishmaniasis.

HLA-DRB1, -DQB1, TNFalpha, TNFbeta, HSP70-2 and HSP70-hom genetic polymorphisms were analyzed in 156 unrelated patients who developed mediterranean visceral leishmaniasis (MVL) due to Leishmania infantum, and 154 unrelated healthy controls, who have got asymptomatic infection with this parasite and were selected on the basis of a positive leishmanin skin test (LST). A significantly reduced frequency of HLA-DR2 was observed among MVL patients (16.1%), compared with controls (26.3%) (relative risk = 0.54; p = 0.04). HLA-DR2/DR13 as well as HLA-DQB1*0201/- genotype frequencies were significantly lower in patients vs controls (relapse rate = 0.17 and 0.46, respectively; p < 0.05). However, using Bonferroni correction, none of these associations remained significant. No association was found, between either the -308 base pair TNFalpha gene polymorphism or the NcoI polymorphism in the first intron of the TNFbeta gene and susceptibility to MVL. Analysis of PstI and NcoI polymorphisms in the coding region of HSP70-2 and HSP70-hom genes, respectively, revealed a significantly higher frequency of homozygotes for the HSP70-2/PstI negative allele, among patients (21.8%) vs controls (12.6%) (relapse rate = 1.94; p = 0.04). Again, this result was not significant after using Bonferroni correction. These results do not support association between susceptibility to MVL and the MHC class II and class III loci analyzed in this study.