Anne Meunier

Environmental risk mapping of canine leishmaniasis in France.

BackgroundCanine leishmaniasis (CanL) is a zoonotic disease caused by Leishmania infantum, a Trypanosomatid protozoan transmitted by phlebotomine sandflies. Leishmaniasis is endemic in southern France, but the influences of environmental and climatic factors on its maintenance and emergence remain poorly understood. From a retrospective database, including all the studies reporting prevalence or incidence of CanL in France between 1965 and 2007, we performed a spatial analysis in order to i) map the reported cases in France, and ii) produce an environment-based map of the areas at risk for CanL. We performed a Principal Component Analysis (PCA) followed by a Hierarchical Ascendant Classification (HAC) to assess if the locations of CanL could be grouped according to environmental variables related to climate, forest cover, and human and dog densities. For each group, the potential distribution of CanL in France was mapped using a species niche modelling approach (Maxent model).ResultsResults revealed the existence of two spatial groups of CanL cases. The first group is located in the Cévennes region (southern Massif Central), at altitudes of 200-1000 m above sea level, characterized by relatively low winter temperatures (1.9°C average), 1042 mm average annual rainfall and much forest cover. The second group is located on the Mediterranean coastal plain, characterized by higher temperatures, lower rainfall and less forest cover. These two groups may correspond to the environments favoured by the two sandfly vectors in France, Phlebotomus ariasi and Phlebotomus perniciosus respectively. Our niche modelling of these two eco-epidemiological patterns was based on environmental variables and led to the first risk map for CanL in France.ConclusionResults show how an ecological approach can help to improve our understanding of the spatial distribution of CanL in France.

Coupling Amperometry and Total Internal Reflection Fluorescence Microscopy at ITO Surfaces for Monitoring Exocytosis of Single Vesicles

Water-soluble hormones and neurotransmitters are packaged in secretory vesicles and secreted into the extracellular medium by exocytosis, a process involving the fusion of the vesicle membrane with the cell membrane. Transport of the secretory vesicles to the cell s periphery, the maturation stages they undergo there to acquire fusion competence, and the factors controlling the fusion process itself (including the dynamics of the fusion pore) are important biological questions that are not fully understood. To elucidate secretory mechanisms at the single-vesicle level, currently only a few analytical methods exist, which can be grouped into electrical or optical recordings. The great advantage of electrical recordings (patch–clamp membrane capacitance and electrochemical amperometry) is their excellent time resolution (ca. tens of microseconds), which allows studies of the dynamics of the fusion pore itself. However, a major disadvantage is the fact that signals appear only after fusion has commenced; that is, the dynamics of the secretory vesicle itself or any labeled regulatory protein prior to the fusion event cannot be detected. In contrast, optical recordings allow secretory vesicles or regulatory proteins to be visualized and tracked prior to their fusion, yet generally they lack the time resolution required to follow the dynamics of the fusion pore (typical time resolution is ca. 100 ms). In addition, depending on the technique, secretion may be probed from different areas of a cell (top or bottom), which makes comparison of the results obtained by different approaches difficult. Because of their complementary nature, it would be a great advance if electrical and optical measurements could be made simultaneously from the same side of a cell at the singlevesicle level. This will enable a comprehensive and precise analysis of the whole exocytotic event, from predocking through fusion steps up to the dynamics of vesicular release. Herein, we report a device based on transparent indium tin oxide (ITO) electrodes, which allows simultaneous total internal reflection fluorescence microscopy (TIRFM) and amperometric measurements (Figure 1). As a proof of concept, the ability of our device in the coupled optical and electrochemical detections of exocytotic events is demonstrated using enterochromaffin BON cells. Amperometry is based on detection at a microelectrode surface positioned near the emitting cell of electroactive vesicular contents that are released into the extracellular medium. With very high temporal resolution and sensitivity, the flux of the vesicular content (released through an initial fusion pore that is only a few nanometers wide) corresponding to an exocytotic event appears as a current spike, which features (frequency, time length, area, magnitude) the dynamics of release from single vesicles. Generally, amperometry involves placing a large collecting electrode near the investigated cell. The whole cell active surface area is covered so the spatial localization of a particular exocytotic event cannot be achieved. Nevertheless, a few studies involving smaller microelectrodes or microelectrode arrays allowed amperometric signals from different releasing sites to be identified, but with a random positioning for the small microelectrode and a spatial resolution necessarily limited by the array dimensions, respectively. Coupling of amperometric and optical recordings would allow precise localization of exocytosis events in space and time. The most widely used optical approach to study exocytosis, TIRFM, is based on the total internal reflection of a laser beam at the glass/water interface, which creates an evanescent field in the aqueous medium whose characteristic decay length (ca. 100 nm) provides a high signal-to-noise ratio and an axial resolution of about 10 nm. When a vesicle fuses with the plasma membrane, its labeled contents are released toward the glass/water interface where the excitation [*] A. Meunier, Dr. R. Fulcrand, Dr. S. Arbault, Dr. M. Guille, Dr. F. Lema tre, Prof. C. Amatore D partement de Chimie, Ecole Normale Sup rieure UMR 8640 (CNRS-ENS-UPMC Univ Paris 06) 24 rue Lhomond, 75005 Paris (France) Fax: (+33)1-4432-3863 E-mail: Christian.Amatore@ens.fr

Indium Tin Oxide devices for amperometric detection of vesicular release by single cells

The microfabrication and successful testing of a series of three ITO (Indium Tin Oxide) microsystems for amperometric detection of cells exocytosis are reported. These microdevices have been optimized in order to simultaneously (i) enhance signal-to-noise ratios, as required electrochemical monitoring, by defining appropriate electrodes geometry and size, and (ii) provide surface conditions which allow cells to be cultured over during one or two days, through apposite deposition of a collagen film. The intrinsic electrochemical quality of the microdevices as well as the effect of different collagen treatments were assessed by investigating the voltammetric responses of two classical redox systems, Ru(NH(3))(6)(3+/2+) and Fe(CN)(6)(3-/4-). This established that a moderate collagen treatment does not incur any significant alteration of voltammetric responses or degradation of the excellent signal-to-noise ratio. Among these three microdevices, the most versatile one involved a configuration in which the ITO microelectrodes were delimited by a microchannel coiled into a spiral. Though providing extremely good electrochemical responses this specific design allowed proper seeding and culture of cells permitting either single cell or cell cluster stimulation and analysis.

Evaluation of the anti-oxidant properties of a SOD-mimic Mn-complex in activated macrophages

SOD-mimics are small complexes that reproduce the activity of superoxide dismutases, natural proteins that catalytically dismutate the superoxide anion. Activated macrophages, which produce ROS and RNS fluxes, constitute a relevant model to challenge antioxidant activity in a cellular context and were used to test a Mn-complex which was shown to efficiently alter the flow of O(2)(-), ONOO(-) and H(2)O(2).