A historical overview of the classification, evolution, and dispersion of Leishmania parasites and sandflies

Background The aim of this study is to describe the major evolutionary historical events among Leishmania, sandflies, and the associated animal reservoirs in detail, in accordance with the geographical evolution of the Earth, which has not been previously discussed on a large scale. Methodology and Principal Findings Leishmania and sandfly classification has always been a controversial matter, and the increasing number of species currently described further complicates this issue. Despite several hypotheses on the origin, evolution, and distribution of Leishmania and sandflies in the Old and New World, no consistent agreement exists regarding dissemination of the actors that play roles in leishmaniasis. For this purpose, we present here three centuries of research on sandflies and Leishmania descriptions, as well as a complete description of Leishmania and sandfly fossils and the emergence date of each Leishmania and sandfly group during different geographical periods, from 550 million years ago until now. We discuss critically the different approaches that were used for Leishmana and sandfly classification and their synonymies, proposing an updated classification for each species of Leishmania and sandfly. We update information on the current distribution and dispersion of different species of Leishmania (53), sandflies (more than 800 at genus or subgenus level), and animal reservoirs in each of the following geographical ecozones: Palearctic, Nearctic, Neotropic, Afrotropical, Oriental, Malagasy, and Australian. We propose an updated list of the potential and proven sandfly vectors for each Leishmania species in the Old and New World. Finally, we address a classical question about digenetic Leishmania evolution: which was the first host, a vertebrate or an invertebrate? Conclusions and Significance We propose an updated view of events that have played important roles in the geographical dispersion of sandflies, in relation to both the Leishmania species they transmit and the animal reservoirs of the parasites

Dielectrophoresis (DEP) based Lab-on-a-chip devices for real-time monitoring of the activity of cytotoxic T-lymphocytes.

Cytotoxic T-lymphocytes (CTL) of cancer patients exhibit the ability to lyse tumor cells and this feature is of great importance for the control of the immune response against tumor onset and progression. Accordingly, the analysis of the cytolytic activity of isolated CTL populations against tumor cells is of great clinical impact, since the isolation of highly cytoxic CTL clones is a complex and long procedure needing of step by step validation of the enrichment achieved. In these experimental protocols, CTL are proposed for the development of tumor immunotherapy. Accordingly, protocols for immunotherapy have been developed for the treatment of melanomas, lymphoblastic leukemia . In this context, realtime monitoring of CTL activity in treated patients might retain prognostic importance. In respect to these issues, dielectrophoresis (DEP) based Lab-on-a-chip technology could represent a very appealing approach, since it allows the manipulation of large numbers of single cells or cell populations. We have employed two dielectrophoresis-based Lab-on-a-chip platforms for the real-time analysis of CTL-mediated cell lysis. One platform (the SmartSlide) displays parallel electrodes and generated cylinder-shaped cages within which CTL and target cells are entrapped. The second platform (the DEP Array) generates spherical DEP cages that can entrap clusters composed of several CTLs and a single (if present) target cell. We demonstrate that DEP-based devices can be used to manipulate CTL clustered to target cells with the aim to develop an efficient methodology to quantify CTL-mediated cytolysis and identification of CTL clusters or clones exhibiting high cytotoxic activity. Target tumor cells were labeled with calcein and their lysis evaluated within the DEP-based platforms. These are the advantages of the proposed approach: (a) no radioactive labeling is required; (b) an high numbers of target cells can be followed in parallel, obtaining statistical information (not possible with the Cr-51 release assay); (c) analysis at the single-cell level is feasible; (d) the protocol is fast and results are reached within 8-12 minutes; (d) the approach is suitable with the use of low numbers of target cells