Approche des transferts de pollution bactérienne dans une crue karstique par l'étude des paramètres physico-chimiques

L'exploitation des systèmes aquifères karstiques est toujours effectuée sous des contraintes liées à leur forte vulnérabilité. Une étude complète d'un épisode de crue de la source du Lez a été réalisée grâce à une coopération entre les Services de Santé et l'Université. Cette étude comporte une observation des paramètres physico-chimiques et bactériologiques sur une période de 15 jours, correspondant à une réponse hydrodynamique impulsionnelle sur la totalité du bassin. Les pas de temps d'échantillonnage varient de 4 à 24 heures.L'interprétation des résultats physico-chimiques met en évidence une disjonction nette entre les variations piézométriques et le passage de différents volumes d'eau. L'écoulement des eaux plus chaudes (16,5 °C) observé lors de l'étiage, eaux d'origine profonde plus chargées chimiquement se poursuit jusque vers le milieu de la décrue pour faire place à l'arrivée d'eaux plus froides correspondant à des infiltrations rapides puis retardées. Les eaux les plus chaudes sont caractérisées par des variations des teneurs en magnésium, les teneurs en calcium restant à peu prés constantes. Inversement; les eaux froides de l'infiltration retardée ont de faibles variations des teneurs en magnésium et sont tracées par une forte augmentation des valeurs en calcium.Le risque sanitaire maximal est lié au début de l'arrivée des eaux froides. Les eaux d'étiage et les eaux profondes sont légèrement contaminées. Les eaux d'infiltration retardée sont peu contaminées. En dehors des zones de perte du réseau hydrographique et des axes d'infiltration rapide, la vulnérabilité de l'aquifère est faible à l'échelle du bassin.The exploitation of karstic aquifer systems for the supply of potable water raises the problem of the evaluation of sanitary risks, in view of their great vulnerability. To supply a great urban tenter, a yield as high as several cubic metres per hour is required, and this magnitude corresponds to that of an underground basin of several km2, so that it is difficult to monitor and protect.Analytical observations from sanitary control platforms on potable water catchments have shown the existence of periods of high bacteriological pollution synchronizing systematically with periods of a rise of water.These bacteriological pollution transfers depend on the general conditions in which the aquifer functions. It seemed interesting, for the prevention of contamination, to examine all these hydrogeologic functions observed during a water rise, using bacteriological parameters.This study was based on observations made during an overall flood period following a prolonged drought throughout the whole basin supplying the city of Montpellier (France). The aim was to carry out a correlation test on the functioning of a karstic aquifer, with regard to :- the variation of the main physico-chemical parameters,- the principal pathogenic germs,- the commonly observed indicators of faecal contamination.This operation, undertaken in close collaboration with the sanitation board and the university authorities, required considerable investment in human, materiat and financial resources.Montpellier, a city with a population of 250 000 inhabitants, is supplied with potable water from the source of the River Lez. The water catchment is-situated upstream of a. major resurgence (12 m3/s during the water rise), tapping water from a principal drainage channel at a depth of 40 metres.The area of the basin feeding the spring was evaluated to be 150 km2 by adjustment to the 450 km2 of Jurassic and Cretaceous outcropping certified limestone, north of Montpellier. Even if the limits of the basin are not precisely defined, some of its parameters are well known. The aquifer consists of different structural zones, almost like independent sub-systems, having in common a rapid drainage network. The response observed at the exurgence is compared globally with the pluviometric signs, as well as with the effects produced in each structural zone, the transfer time, chemistry, temperature, recession, etc.The whole aquifer must be considered under pressure and it is this state of pressure which is probably responsible for the directional flow of the water from the rock matrix and of that circulating in the major karstification areas. The aquifer may be either in a state of injection or drainage and this notion is supported by the chemical and thermal fluctuations at the exurgence point.A one-year experimental programme, based on our knowledge of this aquifer, has been set up in collaboration with forecasters at the National Meteorological Office. All have been on the alert and ready to intervene, equipped with all the necessary staff and apparatus to ensure reliable sampling and analyses. The following procedures were performed :- sampling every 4 hours during the first 48 hours ;- sampling every 12 hours during the following 48 hours.Then until the end of the study, sampling took place every 24 hours with :- 1 bacteriological sample in sterile condition,- 1 sample for physico-chemical analyses in the laboratory,- temperature measurements with a 1/10° mercury thermometer,- resistivity measurements,- pH measurements with two standards,- a limigraphic reading.This period of water rise, hydrologically homogeneous throughout the whote basin in the form of a single impulsive response tasting about 10 days, was characterised by the circulation of two types of water masses : from the rock matrix and resutting from rapid infiltration. The mixture of these two bodies, each with a distinct chemical character, is a function of the state of pressure in the aquifer. At the end of the rise, a third type of water intervenes, that of retarded infiltration.The best criteria for identifying these volumes of water are thermal ones. The most variable elements are magnesium and calcium. Deep waters with a temperature of 16 °5 are characterised by a variation in magnesium, and cold water, by a variation in calcium. The highest value of calcium is related to the passage of water bodies attributed to the influence of retarded infiltration.The geological observations and the processing of results by factorial analyses show a clear distinction between the chemical rise and the piezometric rise. The hydraulic rise point is constituted by water with the saure chemical characteristics as that of the lowest water level. The most important dilution due to rapid transfer in the basin is observed in the middle of the water fait. These synchronized with the highest batteriological pollution rate for most of the elements.This study has provided a global appreciation of the sanitary risks and shows how pollution mechanisms function. Risks are permanent, even during the passage of the deepest water with a long residence time. This phenomenon raises the question of the duration and mode of survive of non encystable bacteria indicators in the karst system. The survival time appeared to be about a year or more.There is always a covariation between the physico-chemical elements and bacteriological pollution where the aquifer is under high pressure and when the water rises or has reached its maximum level, or when the water starts to fall. It is when the level of the water starts to drop that the main peaks of bacteriological pollution are observed. From this moment onwards, the whole basin functions like a drained system and the absence of covariation in the physico-chemical and bacteriological parameters are signs that the flow has become heterogeneous.For sanitation purposes it may be sufficient to monitor temperature and other chemical elements, in order to determine when an additional treatment of the water is required to maintain its potability. An appropriate apparatus is being devised to this end

Attenuation of corneal myofibroblast development through nanoparticle-mediated soluble transforming growth factor-β type II receptor (sTGFβRII) gene transfer

Purpose: To explore (i) the potential of polyethylenimine (PEI)-DNA nanoparticles as a vector for delivering genes into human corneal fibroblasts, and (ii) whether the nanoparticle-mediated soluble extracellular domain of the transforming growth factor–β type II receptor (sTGFβRII) gene therapy could be used to reduce myofibroblasts and fibrosis in the cornea using an in vitro model. Methods: PEI-DNA nanoparticles were prepared at a nitrogen-to-phosphate ratio of 30 by mixing linear PEI and a plasmid encoding sTGFβRII conjugated to the fragment crystallizable (Fc) portion of human immunoglobulin. The PEI-DNA polyplex formation was confirmed through gel retardation assay. Human corneal fibroblasts (HCFs) were generated from donor corneas; myofibroblasts and fibrosis were induced with TGFβ1 (1 ng/ml) stimulation employing serum-free conditions. The sTGFβRII conjugated to the Fc portion of human immunoglobulin gene was introduced into HCF using either PEI-DNA nanoparticles or Lipofectamine. Suitable negative and positive controls to compare selected nanoparticle and therapeutic gene efficiency were included. Delivered gene copies and mRNA (mRNA) expression were quantified with real-time quantitative PCR (qPCR) and protein with enzyme-linked immunosorbent assay (ELISA). The changes in fibrosis parameters were quantified by measuring fibrosis marker α-smooth muscle actin (SMA) mRNA and protein levels with qPCR, immunostaining, and immunoblotting. Cytotoxicity was determined using cellular viability, proliferation, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Results: PEI readily bound to plasmids to form nanoparticular polyplexes and exhibited much greater transfection efficiency (p<0.01) than the commercial reagent Lipofectamine. The PEI-DNA-treated cultures showed 4.5×10[superscript 4] plasmid copies/µg DNA in real-time qPCR and 7,030±87 pg/ml sTGFβRII protein in ELISA analyses, whereas Lipofectamine-transfected cultures demonstrated 1.9×10[superscript 3] gene copies/µg DNA and 1,640±100 pg/ml sTGFβRII protein during these assays. The PEI-mediated sTGFβRII delivery remarkably attenuated TGFβ1-induced transdifferentiation of corneal fibroblasts to myofibroblasts in cultures, as indicated by threefold lower levels of SMA mRNA (p<0.01) and significant inhibition of SMA protein (up to 96±3%; p<0.001 compared to no-gene-delivered cultures) in immunocytochemical staining and immunoblotting. The nanoparticle-mediated delivery of sTGFβRII showed significantly better antifibrotic effects than the Lipofectamine under similar experimental conditions. However, the inhibition of myofibroblast in HCF cultures by sTGFβRII overexpression by either method was significantly higher than the naked vector transfection. Furthermore, PEI- or Lipofectamine-mediated sTGFβRII delivery into HCF did not alter cellular proliferation or phenotype at 12 and 24 h post-treatment. Nanoparticles treated with HCF showed more than 90% cellular viability and very low cell death (2–6 TUNEL+ cells), suggesting that the tested doses of PEI-nanoparticles do not induce significant cell death. Conclusions: This study demonstrated that PEI-DNA nanoparticles are an attractive vector for the development of nonviral corneal gene therapy approaches and that the sTGFβRII gene delivery into keratocytes could be used to control corneal fibrosis in vivo.National Institutes of Health (U.S.) (RO1EB000244

BMP7 Gene Transfer via Gold Nanoparticles into Stroma Inhibits Corneal Fibrosis In Vivo

This study examined the effects of BMP7 gene transfer on corneal wound healing and fibrosis inhibition in vivo using a rabbit model. Corneal haze in rabbits was produced with the excimer laser performing -9 diopters photorefractive keratectomy. BMP7 gene was introduced into rabbit keratocytes by polyethylimine-conjugated gold nanoparticles (PEI2-GNPs) transfection solution single 5-minute topical application on the eye. Corneal haze and ocular health in live animals was gauged with stereo- and slit-lamp biomicroscopy. The levels of fibrosis [α-smooth muscle actin (αSMA), F-actin and fibronectin], immune reaction (CD11b and F4/80), keratocyte apoptosis (TUNEL), calcification (alizarin red, vonKossa and osteocalcin), and delivered-BMP7 gene expression in corneal tissues were quantified with immunofluorescence, western blotting and/or real-time PCR. Human corneal fibroblasts (HCF) and in vitro experiments were used to characterize the molecular mechanism mediating BMP7’s anti-fibrosis effects. PEI2-GNPs showed substantial BMP7 gene delivery into rabbit keratocytes in vivo (2×10[superscript 4] gene copies/ug DNA). Localized BMP7 gene therapy showed a significant corneal haze decrease (1.68±0.31 compared to 3.2±0.43 in control corneas; p88%; p<0.0001), and immunoblotting of BMP7-transefected HCFs grown in the presence of TGFβ demonstrated significantly enhanced pSmad-1/5/8 (95%; p<0.001) and Smad6 (53%, p<0.001), and decreased αSMA (78%; p<0.001) protein levels. These results suggest that localized BMP7 gene delivery in rabbit cornea modulates wound healing and inhibits fibrosis in vivo by counter balancing TGFβ1-mediated profibrotic Smad signaling.National Institutes of Health (U.S.) (RO1EB000244)Mason Eye Institute (Research to Prevent Blindness Unrestricted Grant

Global Public Health Security

National public health institutes will play a key role in implementation of the revised International Health Regulations