Phytochemical composition and antioxidant activity of medicinal plants collected from the Tunisian flora.

Polyphenols, flavonoids and condensed tannins contents, as well as the antioxidant activity of the ethanolic and aqueous extracts obtained from aerial parts of 10 wild Tunisian plants, have been determined. Extracts showed appreciable levels of polyphenols and flavonoids, which reached 215.16 mg GAE g-1 DW in Lavandula stoechas ethanolic extract, and 49.12 mg RE g-1 DW in Thapsia garganica aqueous extract. The majority of tested extracts exhibited low total condensed tannins content, except for Rhus tripartitum and Periploca laevigata. The antioxidant activity tests showed great activity, especially for R. tripartitum and Lavandula multifida (IC50 = 5.16 and 5.1 µg mL-1, respectively). Canonical Correspondence Analysis revealed clear groupings of species according to the solvent used.

The role of Toll-like receptor 9 in gynecologic cancer.

Toll-like receptor 9 (TLR9) plays a major role in the fight against DNA viruses infections. Despite its antitumor properties, inappropriate activation of TLR9 during chronic inflammation may cause the activation of transcription factors inducing pro-cancerous activities. Thus, the relationship between TLR9 and cancer remains highly confrontational especially in gynecological cancers and cervical cancer induced by viruses. In this review, we focus on the beneficial and detrimental role of TLR9 in gynecological carcinogenesis. TLR9 contributes to tumor regression by inducing cytotoxic T cell response (CTL), reducing the numbers of myeloid-derived suppressor cells (MDSCs), the tumor-associated macrophages (TAMs) and the regulatory T cells (T regs). It can however, also promote tumor progression and invasiveness of cervical tissue. Therefore, the dichotomous role of TLR9 needs to be carefully investigated in the setting of neoplastic disease.

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.

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.

Immunochromatographic rK39 strip test in the serodiagnosis of visceral leishmaniasis in Tunisia.

The performance of the rK39 strip test in the diagnosis of Tunisian visceral leishmaniasis (VL) was evaluated and compared with that of immunofluorescent antibody test (IFAT). A total of 929 sera, including 574 from VL patients, 54 from cutaneous leishmaniasis (CL) patients, 42 from patients with other protozoan diseases, 152 from patients with non-parasitic diseases and 107 from healthy controls, were used in the study. The sensitivity and specificity of the rK39 strip test were 87.1 and 94.4%, respectively. Sixteen CL sera showed positive results, suggesting that the rK39 strip test is not restricted to Leishmania donovani complex detection. IFAT was comparatively more sensitive (98.9%) but slightly less specific (90.7%). Despite cross-reactivity shown by CL sera, the rK39 strip test can be recommended for the routine diagnosis of VL in Tunisia, as VL and CL are distinct clinical entities.

[A variant of DDRT-PCR using anchored mini-exon primers for identification of differentially expressed sequences in Leishmania infantum]. / Une variante du dDRT-PCR utilisant des amorces mini-exon ancrées pour l'identification de séquences différentiellement exprimées chez leishmania infantum.

Leishmania infantum (L.i) is responsible for visceral (VL) or cutaneous (CL) leishmaniasis. Previous studies done in Honduras by differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) failed to demonstrate differences in expression profiles among L. infantum VL and CL parasites. For purpose of comparing expression among L. infantum isolates in Tunisia, a variant of this technique adapted from a commercial kit was developed involving pairs of random and anchored mini-exon primers for isolation and identification of differentially displayed cDNAs. To assess the efficiency of this variant, 34 pairs were applied to 2 consecutive dilutions of cDNAs from promastigotes at end of in vitro exponential growth of 2 visceral (LV50) and cutaneous (DREP14) isolates from Tunisia, thus increasing chance for observing differences among the cDNAs. Profiles were compared and analyzed as regards number and phenotype of bands displayed in 4 types of highly similar amplification profiles among the 2 cDNAs; 26 primer pair combinations generated in total 6.8% differentially displayed bands that had variable intensities or were present/absent, in comparable proportions in the 2 isolates. Analysis further demonstrated differences in amplification efficiency of some primers, emphasizing on qualitative and quantitative impact of relative proximity of the priming sites. Nine present/absent bands were cloned, sequenced and analyzed in silico. Mismatches at priming sites seem to underlie amplification of such bands. Only five products could be referred to annotated gene. Among the genes identified, we list histone H4, largely known to be differentially expressed among L.i stages, and "NTF2-like" for which overexpression in one cDNA was here confirmed. To conclude, the variant developed could be used further in Leishmania expression analysis with appropriate cautions about false positives.

[Differentiation among cutaneous Leishmania species upon amplification of a sequence of dipeptidyl peptidase III encoding gene]. / Différenciation des espèces responsables de leishmaniose cutanée par une amplification PCR du gène codant pour la dipeptidyl peptidase III.

Leishmaniasis are a group of vector-born, parasitic diseases caused by protozoan of the Leishmania genus, that includes visceral or cutaneous forms. Cutaneous leishmaniasis (CL) refers to a group of diseases because of the variability of clinical manifestations, caused by a large number of Leishmania species. In Tunisia, three different forms of CL are encountered, having different causal agents L. infantum, L. major and L. tropica. For the purpose of this study, we assessed the potential of polymorphic sites in dipeptidyl peptidase III (DPP III) encoding gene to differentiate among Leishmania species encountered in Tunisia. A pair of forward and reverse primers amplifying a 664 bp DPP III sequence were designed in regions including 2 mutations in the forward primer and 1 in the reverse, and were used to amplify DNA from diverse species of Leishmania parasites including L. infantum, L. major, L. tropica, L. donovani, L. chagasi, L. arabica, L. aethiopica and L. tarentolae. Amplification was positive for all tested Leishmania species except for L. infantum, L. chagasi, L. archibaldi, L. donovani and L. tarentolae. In case of cutaneous Leishmania species encountered in Tunisia, amplification was positive for both L. tropica and L. major and negative in case of L. infantum. This ability to differentiate L. infantum from L. tropica/L. major constitutes a first step in the taxonomy of cutaneous species prevalent in Tunisia.

Identification of Leishmania donovani as a cause of cutaneous leishmaniasis in Sudan.

Eight patients with cutaneous ulcers were referred to the Institute of Endemic Diseases, Khartoum, Sudan, from June 2000 to March 2002 for the diagnosis of suspected cutaneous leishmaniasis (CL). Diagnosis was confirmed parasitologically by both positive Giemsa-stained smears and successful culture of Leishmania promastigotes in NNN medium. The eight parasite isolates were shown to belong to the Leishmania donovani complex by kDNA PCR. Isoenzyme typing of three isolates revealed that they were identical to the L. donovani MON-82 reference strain, and the gp63 PCR-RFLP profile showed similar patterns to a reference strain of MON-82. CL is endemic in most regions of Sudan and has been reported previously as being caused by L. major MON-74. The results of this study suggest that L. donovani is also a cause of CL in Sudan and that further study of isolates from Sudanese patients with cutaneous ulcers is warranted to ascertain whether L. donovani or L. major is the causative agent.

[Description of the female of Phlebotomus (Larroussius) Chadlii, Rioux, Juminer & Gibily, 1966 (Diptera: Psychodidae). From an example captured from the area of Kef (Tunisia)]. / Description de la femelle de Phlebotomus (Larroussius) Chadlii Rioux, Juminer et Gibily, 1966 (Diptera : Psychodidae). D'après un exemplaire capturé aux environs du Kef (Tunisie).

The female of Phlebotomus chadlii Rioux, Jumminer & Gibily, 1966 is described and illustrated for the first time from a specimen collected in El Kef region, northwest Tunisia. It was distinguished from P. ariasi by several characters of the spermathecae: 1) the enlarged portion of P. chadlii spermathecae duct appears smooth and better developed than that of P. ariasi; 2) in P. chadlii, this part comprises three quarters of the duct whereas, in P. ariasi, it covers only the half; 3) the spermathecae neck of P. chadlii is shorter than that of P. ariasi. The duct base is compatible with the large aedeagus size of P. chadlii male. Besides, the assignment of this female to the species P. chadlii is supported by: 1) the presence of males in the same area, over the last three years; 2) the total absence in this area of P. ariasi; 3) the concomitant presence, in the same trap station, of the described female with P. chadlii males.

Uncommon clinical presentations of cutaneous leishmaniasis in Sudan.

Cutaneous leishmaniasis in Sudan is caused by Leishmania major zymodeme LON1. Self-healing usually occurs within 1 year but occasionally its duration is prolonged and treatment is required. The clinical forms are ulcers, nodules and noduloulcerative lesions. Here we describe seven patients with uncommon lesions that were difficult to recognize as Leishmania infections. These included mycetoma-like lesions, lesions that resembled L. tropica infection and others. One HIV/AIDS patient had Kaposi's sarcoma with Leishmania parasites in the Kaposi lesions. Most of these uncommon clinical forms were difficult to treat. The diagnosis depended on a high degree of suspicion and the demonstration of parasites in smears or culture. PCR was used to characterize parasites from the patients described here. Leishmania major was found by kDNA PCR in all patients, except one, who had a leishmanioma due to L. donovani. In three patients, including one with a L. tropica like-lesion, the parasites were confirmed as L. major by gp63 PCR-RFLP.

Molecular analyses of Old World Leishmania RAPD markers and development of a PCR assay selective for parasites of the L. donovani species Complex.

Three amplicons, appearing in a species-specific manner on the electrophoregrams of RAPD reactions that were obtained with primer OPA1, OPA1-800, OPA1- 900, and OPA1-1200, are analyzed in this study. The study revealed that each of these products is composed of one Leishmania DNA band, taxonomically conserved among the different Old World species studied. Subsequently, only the electrophoretic position of the RAPD products can be considered species-specific. In addition, sequence data, genomic organization, and chromosomal location have proved that these fragments are different and physically independent. However, they possess common features related to the presence of different kinds of short DNA repeats, more particularly microsatellites and a CCCTTC motive, corresponding to the 3' half of the OPA1 primer. These results suggest that the OPA1 primer has initiated amplification from different priming sites, having a species-specific location. This corresponds to sequence micro-heterogeneity of DNA fragments present within the different species and leading eventually to a selective amplification of different RAPD products. This characteristic has been used to develop an original selective PCR test based on the sequence of the OPA1-800 product, in which only DNAs from the L. donovani species complex are amplified. Restriction site polymorphisms and sequence variations are identified within the PCR fragment amplified from these parasite DNAs. In fact, the OPA1-800 fragment proved to be a useful DNA marker either as a DNA probe or as a target for PCR-based assays. This tool can therefore be recommended for the control of Old World Leishmania parasites, such as species discrimination, molecular tracking of isolates, or study of polymorphisms within the L. donovani species complex. Moreover, the molecular bases underlying the amplification of the RAPD fragments studied correspond to mechanisms already described. Although they do not account for the amplification of all Leishmania RAPD products, such mechanisms stress some of the pitfalls of the technique, which need to be taken into consideration. We have identified at least misleading observations of DNA bands amplified in a species-specific manner, in spite of their presence in the genome of the other taxa, and relatedness between bands within the amplification profiles. Therefore, recommendations for careful interpretation of RAPD data in population genetics or phylogenetic analyses are reiterated. Molecular analyses are essential to validate conclusions.

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.

Identification of Lebanese dermotropic putative Leishmania archibaldi isolates by gp63 PCR-RFLP.

Leishmania stocks isolated from cutaneous lesions in Lebanon were characterized by PCR methods. The stocks were typed as putative L. (L.) archibaldi (gp63 PCR-RFLP), belonging to 2 different genotypes (PCR-based schizodeme analysis). This constitutes the first report on the presence of L. (L.) archibaldi in the Middle East.

[Evaluation of two DNA probes specific for Leishmania infantum in the diagnosis of canine leishmaniasis]. / Evaluation de deux sondes ADN spécifiques de Leishmania infantum dans le diagnostic de la leishmaniose canine.

This study reports on the evaluation of two L. infantum specific DNA probes for the diagnosis of canine leishmaniasis. The probes presented very satisfying performances in terms of specificity (100%) and predictive value of the positive result (100%). However, their sensitivity (35.3%) and the clinical complexity of canine infections render their use difficult in epidemiological surveys of visceral leishmaniasis aiming at measuring the prevalence of the dog infection by L. infantum. The sensitivity of these tools has improved (66.7%) when dogs presenting patent leishmaniasis were considered. Such probes constitute appropriate tools to confirm suspected cases of leishmaniasis. Unlike the classical parasitological and serological tools, this kind of tools allows a concomitant detection and identification of the causative agent. Therefore, despite their low sensitivity, these probes can still be of importance in epidemiological investigations.

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.

[Epidemiologic and parasitologic data concerning sporadic cutaneous leishmaniasis in northern Tunisia]. / Données épidémiologiques et parasitologiques concernant la leishmaniose cutanée sporadique du nord tunisien.

This study refers to 23 patients presenting with the sporadic forms of cutaneous leishmaniasis encountered in northern most humid parts of Tunisia. Culture inoculation for parasitic isolation was processed using two media: the classical NNN and a rabbit serum based medium (SLC). Cultures were positive in 17 cases with SLC medium and 13 cases with NNN medium. Eight isolates were typed using 15 isoenzymes systems. Six isolates were identified as Leishmania infantum MON-24 which confirms the crucial role of this zymodeme in causing this form of cutaneous leishmaniasis. The other two isolates were identified as Leishmania infantum MON-1, which is the principal agent of visceral leishmaniasis in the Mediterranean area.

Leishmania infantum species-specific kDNA probes: isolation and evaluation.

The study refers to the isolation of specific DNA probes to the parasite species Leishmania (L) infantum according to different strategies using recombinant minicircles isolated from L. infantum kinetoplast DNAs. A first probe was identified following a classical procedure. One mini-circle selected for strong reactivity to L. infantum total DNA was used to identify specific subfragments to this species among which the 95bp fragment, 3B8HaeIII-2 was selected. For the obtention of the second probe, a strategy based on sequential screenings for specificity and sensitivity was applied. This allowed identification of a set of minicircles showing an increased specificity to L. infantum as compared to other species, and an increased sensitivity of reaction as compared to the other minicircles. Subclonings and screenings allowed a final selection of a 137bp-minicircle fragment: 3E9HaeIII-12. Reactivities of the 2 probes were assessed on a panel of total DNAs and promastigotes from 74 isolates pertaining to 9 species encountered in the Old World. Parasites isolated in Tunisia from different foci, different hosts after different transmission seasons were included. Hybridizations have shown the exquisite specificity of these probes to L. infantum in this country. Probe 3E9HaeIII-12 was found to be the more sensitive where down to 10 ng of total DNA and 10(3) promastigotes could be detected. From this study and as compared to data provided in the literature, the second procedure allowed at least 10-fold increase in sensitivity.

[Validation of DNA tools in the identification of Leishmania sp. parasites in Algeria]. / Validation d'outils ADN dans l'identification de parasites Leishmania sp. en Algérie.

The present study aimed at homogenizing the use of DNA tools for Leishmania parasite characterization in two endemic countries, Algeria and Tunisia. Two genomic DNA probes, pDK10 and pDK20, previously developped in Tunisia, were here applied to a collection of 41 isolates obtained from Algerian patients having cutaneous or visceral leishmaniases. These DNA tools allowed to discriminate among and to identify causal agents of cutaneous leishmaniasis, L. infantum and L. major. Apart from the pDK20--hybridization pattern obtained usually for the species L. infantum, new hybridization patterns were identified for isolates obtained from both visceral and cutaneous leishmaniases patients. Use of DNA probes in complement to isoenzyme typing offers interesting propects for a better description of transmission cycles.