New method for screening anti-Leishmania compounds in plants extracts by HPTLC-bioautography.

Leishmania genus is responsible for leishmaniasis, a group of diseases affecting 12 million people in the tropical and subtropical zone. Currently, the few drugs that are available to treat this disease are expensive and cause many side effects. Searching for new therapeutics from plant species seems to be a promising path. This work proposes an original HPTLC test against parasites, in particular on Leishmania infantum, to screen new molecules from plant extracts. The technique uses protozoa transformed to express the luciferase gene to observe the bioautogram in bioluminescence. We have developed two different test protocols based on the two dimorphic stages of the parasite. The free promastigote stage, and an intracellular stage parasitizing macrophage cells called the amastigote stage. These two stages only survive under extremely different conditions which required the development of two very different test protocols. For the promastigote free stage of the protozoa, the direct bioautography technique was chosen while for the intracellular amastigote stage, bioautography by immersion (agar overlay) was required. Amphotericine B was chosen as the reference compound for this assay. The development of each of these two tests made it possible to clearly detect areas of activity on the bioautogram, allowing a rapid and inexpensive screening of the antiparasitic properties of molecules in natural extracts.

Identification of adipocytes as target cells for Leishmania infantum parasites.

Leishmania infantum is the causative agent of visceral leishmaniasis transmitted by the bite of female sand flies. According to the WHO, the estimated annual incidence of leishmaniasis is one million new cases, resulting in 30,000 deaths per year. The recommended drugs for treating leishmaniasis include Amphotericin B. But over the course of the years, several cases of relapses have been documented. These relapses cast doubt on the efficiency of actual treatments and raise the question of potential persistence sites. Indeed, Leishmania has the ability to persist in humans for long periods of time and even after successful treatment. Several potential persistence sites have already been identified and named as safe targets. As adipose tissue has been proposed as a sanctuary of persistence for several pathogens, we investigated whether Leishmania infantum could be found in this tissue. We demonstrated both in cell cultures and in vivo that Leishmania infantum was able to infect adipocytes. Altogether our results suggest adipocytes as a 'safe target' for Leishmania infantum parasites.

Escherichia coli Rho GTPase-activating toxin CNF1 mediates NLRP3 inflammasome activation via p21-activated kinases-1/2 during bacteraemia in mice.

Inflammasomes are signalling platforms that are assembled in response to infection or sterile inflammation by cytosolic pattern recognition receptors. The consequent inflammasome-triggered caspase-1 activation is critical for the host defence against pathogens. During infection, NLRP3, which is a pattern recognition receptor that is also known as cryopyrin, triggers the assembly of the inflammasome-activating caspase-1 through the recruitment of ASC and Nek7. The activation of the NLRP3 inflammasome is tightly controlled both transcriptionally and post-translationally. Despite the importance of the NLRP3 inflammasome regulation in autoinflammatory and infectious diseases, little is known about the mechanism controlling the activation of NLRP3 and the upstream signalling that regulates the NLRP3 inflammasome assembly. We have previously shown that the Rho-GTPase-activating toxin from Escherichia coli cytotoxic necrotizing factor-1 (CNF1) activates caspase-1, but the upstream mechanism is unclear. Here, we provide evidence of the role of the NLRP3 inflammasome in sensing the activity of bacterial toxins and virulence factors that activate host Rho GTPases. We demonstrate that this activation relies on the monitoring of the toxin's activity on the Rho GTPase Rac2. We also show that the NLRP3 inflammasome is activated by a signalling cascade that involves the p21-activated kinases 1 and 2 (Pak1/2) and the Pak1-mediated phosphorylation of Thr 659 of NLRP3, which is necessary for the NLRP3-Nek7 interaction, inflammasome activation and IL-1ß cytokine maturation. Furthermore, inhibition of the Pak-NLRP3 axis decreases the bacterial clearance of CNF1-expressing UTI89 E. coli during bacteraemia in mice. Taken together, our results establish that Pak1 and Pak2 are critical regulators of the NLRP3 inflammasome and reveal the role of the Pak-NLRP3 signalling axis in vivo during bacteraemia in mice.

Leishmania infection: Misdiagnosis as cancer and tumor-promoting potential.

Given the prevalence of cancer and leishmaniasis worldwide, the presence of these two pathologies in the same tissue sample may be merely fortuitous. The clinical outcome of both diseases is under the control of innate and adaptive immunity, and in both cases these progressive diseases are characterized by an impaired host Th1 response. As a consequence, the Th2 cytokine microenvironment occurring in progressive leishmaniasis may potentially promote tumor cell proliferation and vice versa. On the other hand, clinical aspects of subclinical cutaneous or visceral leishmaniasis sometimes closely resemble those observed in various neoplasms thus leading to misdiagnosis. In this review, we present recent findings on the association between leishmaniasis and malignant disorders. Our review includes HIV positive, HIV negative subjects and patients whose HIV status has not been established. Leishmaniasis mimicking a malignant disorder was confirmed and extended to unreported neoplastic disorders including squamous cell carcinoma, T-cell and B-cell lymphoma, oral and intranasal tumors and granulomas. Thus, leishmaniasis should be considered in the differential diagnosis and course of various cancers in Leishmania endemic areas or in patients with travel history to these areas. We also listed recent reports showing that Leishmania can promote cancer development in immunocompromised as well as in immunocompetent patients. The potential mechanisms supporting this promoting effect are discussed.