Efficacité des méthodes d'adsorption-élution utilisant la poudre ou la laine de verre pour la concentration des virus dans les effluents de stations d'épuration

Pour rechercher les virus dans les eaux usées traitées, une nouvelle méthode d'adsorption-élution sur laine de verre a été appliquée comparativement à la méthode d'adsorption-élution sur poudre de verre. Lorsque la technique de concentration sur laine de verre est utilisée, c'est dans les 25 premiers ml de l'éluat que la majorité des virus poliomyélitique est retrouvé (89 à 94 %). La comparaison des méthodes de concentration des virus indigènes à partir d'échantillons d'effluents provenant de deux stations d'épuration biologique de la Côte d'Azur (Cagnes-sur-Mer et Nice), a mis en évidence la supériorité de cette nouvelle méthode : les taux de positivité ont été respectivement de 85 % vs 38 % pour l'effluent de Cagnes-sur-Mer et 100 % vs 44 % pour l'effluent de Nice. De même, les titres en virus indigènes après concentration ont varié de 0 à 250 NPPUC/l pour la méthode sur laine de verre contre 0 à 25,5 NPPUC/l pour la méthode sur poudre de verre. La différence constatée entre les méthodes est statistiquement significative après analyse de variance (p = 0,0119 pour l'effluent de Cagnes-sur-mer et p < 0,0001 pour l'effluent de Nice). De plus, la technique sur laine de verre ne nécessite ni l'abaissement du pH, ni le changement de la composition ionique de l'échantillon d'eau à analyser.Biological treatment of sewage in waste water plants does not allow elimination of the whole of the microbial load. Discharge of the treated sewage results in viral pollution of river, lakes and seas, a potential hazard for the health that has to be monitored. The amont of virus in waste water beeing low, concentration from the samples brought to the laboratory is rendered necessary. The aim of this study was to evaluate the efficacy of a new adsorption-elution method on glass wool to recover indigenous viruses from effluents of the cities of Cagnes and Nice (Alpes-Maritimes, France). In order to evaluate its efficiency we compared it to the regular adsorption-elution method on glass powder. As a preliminary we determined upon artificially contaminated 5 liter waste waters samples what detection of virus could be performed only in the first 25 ml of the 100 ml eluate, as in the glass powder concentration method. Results show chat virus titers found in that first fraction of eluate were close to those in the total sample. Thus from 3 samples containing 1.60 108 MPNCU/5 l, 1.96 107 MPNCU/5 l and 4.32 104 MPNCU/5 l we found in that first fraction respectively 1.50 104 MPNCU/5 l (94 %), 1.80 107 MPNCU/5 l (92 %) and 3.85 104 MPNCU/5 l (89 %); these recovery rates are not significantly different by comparison of confidence limits. The glass powder method, necessitates preliminary treatment of the sample : acidification to pH 3.5 and adjunction of AICI3 at a final concentration of 5.10-4 M. After flowing the acidified sample through 100 g of borosilicated glass powder at a rate of 10 l/10 min inside a decantation ampulla. Then adsorbed virus may be eluted from the sedimentated glass powder with 100 ml of borate buffer containing 3 % beet extract, pH9 : the first 25 ml were collected into a flask containing 2.5 ml of a mixture of antibacterial and antifungal antibiotics. For the glass wool adsorption method, a 19 cm3 cartridge was packed with 5 g sodocalcic glass wool at a 0.4 g/cm3 density and rinced sequentially with : 10 ml 1N HCl, 10 ml deionised water, 10 ml 1N NaOH and lastly 40 ml deionised water. It was balanced with 200 ml deionised water. The sample, was pumped at a flow rate ca 10 l/h. Enumeration of viruses was performed by inoculating 40 microplate wells containing KB cells, and performing 3 passages 5 days each, after which the number of wells presenting with CPE was determined. This characteristic number allowed calculation of the most probable number of cytopathic units (MPNCU) with the 95 % confidence limit. The Box and Cox analysis of transformation was applied to the data. Since the calculated value of λ approximated zero (λ = - 0.29 for the Cagnes effluent and λ = - 0,062 for the Nice effluent), transformation of the gross data into logarithm was justified. To allow this transformation, the zero had to be substituted for by a value equal to half the limit sensitivity of the method (I well out of 40), i.e. 0.5. Distribution of the data being roughly log-normal, it was then possible to compare the results of the two methods by two-way analysis of variance, cross classification, without replica. The test for factor method was calculated according to the interaction since this factor is fixed. Overall it appeared that all 31 10-liter samples analysed contained viruses when results from bath methods were combined. Still no single method allowed virus recovery in a 100 % of cases, however the glass wool adsorption method found viruses in 29/31 vs 13/31 with the glass powder method. The new method detected virus in 11/13 (85 %) samples from Cagnes waste waters as well as in 18/18 (100 %) from Nice. Quantitative analysis of the viral titers indicates that, titers were higher following the glass wool adsorption method than following the glass powder adsorption method in 11/13 samples from Cagnes treatment plant and in 17/18 from Nice. Thus virus concentrations varied between 0 and 250 MPNCU/l (MGT= 4.6 MPNCU/l) for the Cagnes effluent and between 2 and 60 MPNCU/l (MGT= 7.5 MPNCU/I) for the Nice effluent. For the same samples virus concentrations obtained following glass powder adsorption method varied between 0 and 8.5 MPNCU/l (MGT 0.9 MPNCU/l) for the Cagnes effluent and between 0 and 25.5 MPNCU/l (MOT= 1.3 MPNCU/l1) for the Nice effluent. This difference is statistically significant (p = 0.0119 for the Cagnes effluent and p < 0.0001 for the Nice effluent). Furthermore, when taking into account the origin of the waters analysed, comparison between observed F0.95 (7.94 for the Cagnes waters and 45.78 for the Nice waters) and theoretical F0.95 (4.75 for the Cagnes waters and 4.45 for the Nice waters) leads to the rejection of the hypothesis of identity of the two methods. The discordances observed are an illustration of the fact that concluding to the absence of viruses in a given sample is a matter of method and should be interpreted with prudence. A few drawbacks inherent to the glass powder adsorption method may explain its poorer efficiency : the necessary acidification of the sample to pH 3.5 may be fatal to a proportion of virions; also the flow rate necessary to maintain the fluid layer of glass powder in suspension during the adsorption step is 6 fold higher than that required in the glass wool method (60 l/h vs 10 l/h). Finally the nature of the adsorbing material, sodocalcic vs borosilicated, may be determinent. We can conclude from the present comparative study, to the statistically significant superiority of the glass wool method for virus concentration from treated waste waters

Etude d'un réseau à très faible teneur en matière organique biodégradable : le réseau de la ville de Nice

La Ville de Nice bénéficie de deux ressources en eau d'excellente qualité, la Vésubie et la nappe alluviale du Var, qui se caractérisent par leur très faible teneur en matière organique biodégradable. La Compagnie Générale des Eaux, gestionnaire de son alimentation en eau potable, applique depuis sa mise au point au début du siècle le procédé de désinfection par l'ozone et distribue une eau sans ajouter de chlore.Un diagnostic complet de la qualité microbiologique et sanitaire de ce réseau non chloré et sans Carbone Organique Dissous Biodégradable (CODB) a été entrepris.Le suivi de la qualité de l'eau dans le réseau a consisté en des campagnes de prélèvements à partir de plusieurs points d'observation répartis sur l'ensemble du réseau. Ce suivi a permis de confirmer la bonne qualité de l'eau distribuée et le maintien de cette qualité tout au long de son transport dans les canalisations. Le CODB est absent sur l'ensemble des points confirmant l'absence de relargage et de contamination pendant le transport.La récupération des dépôts présents dans les canalisations n'a pas révélé de colonisation du réseau par des micro-organismes supérieurs. La quantité de biofilm présente sur les parois des canalisations a été évaluée en suivant la colonisation de pastilles de matériau immergées dans les réservoirs.Ce diagnostic complet de l'état microbiologique et sanitaire du réseau de Nice montre que l'absence de chloration n'entraîne pas de dégradation de la qualité bactériologique de l'eau et que c'est essentiellement l'absence de matière organique biodégradable qui limite la croissance bactérienne et qui contribue au maintien de la qualité de l'eau dans le réseau de distribution.Within the more general scope of studies aiming at a better knowledge of the mechanisms responsible for biological biodegradation in water systems, it was interesting to learn the microbiological behaviour of a drinking water network supplied with water free of biodegradable organics. The case in point is the town of Nice which enjoys excellent quality water ressources, to the extent that the Compagnie Générale des Eaux, managers of the municipal water utility, were able to apply ozonation as a disinfectant when the technique first appeared at the beginning of the century, and supply chlorine free water.The Municipal Services of the Town of Nice, the Nice Health Laboratory and the Compagnie Générale des Eaux bave therefore combined forces to carry out a study on the development of water quality in the supply network in such an animal environment. The objective is to make a complete diagnosis of the microbiological and hygienic quality of a chlorine-free network which would confirm the excellent quality of the distributed water and serve as a reference within the scope of general research on the removal of biodegradable organics for the supply of non chlorinated water.The study consisted of monitoring three supply systems fed by three different water works :- the Super Rimiez plant treating water from the river Vésubie in the following stages : flocculation, primary clarification, sand filtration and virucidal ozonation;-the Jean Moreno plant : water pumped from the alluvion layers of the Var and ozonated;-the Prairies plant : pumping and temporary chlorination of the Var aquifer with chlorine dioxyde.In order to take seasonal variations info account, our study lasted for a whole year, with sampling campaigns every two weeks on 20 observation points along the network, and on the raw water treated in three production plants. The physico-chemical and microbiological characteristics of the water samples were analyzed.Samples were collected at a high flow rate from fire hydrants near the observation points. The sediments were gathered in a plankton net and analyzed to detect the presence of possible animalcules.The immersion of PVC coupons in two reservoirs, fed respectively with the two types of water, enabled the growth kinetics of fixed bacteria to be followed, and the quantifies of fixed biomass at steady state to be compared.It was possible, by monitoring the chloride content (on average 25 mg Cl¯/l in Var water compared with 5 mg Cl¯/l in Vésubie water), to identify the precise influence of the two water resources.Total bacteria measured by epilluorescence are not very high compared with networks in other large towns. It will be noted, however, that the figure is higher in the network supplied from the Vésubie than in those treating water from the Var aquifer. Bacterial regrowth in circulating water is minor, showing that the influence of bacteria released by the biofilm installed on the pipe walls is minimized. This biofilm is present just the same, as is shown by the results of colonization an the immersed coupons in reservoirs.The quantity of fixed bacteria at steady state is different according to the type of water but, in both cases, it was lower than the figure obtained with water loaded with biodegradable organics. The total plate count remains low on the whole, even at end sections of the network. The presence at fecal bacteria was only observed at a few points during some of the studies and the Nice network, on the whole, is of excellent quality judged by the very few fecal contamination indicators found.The presence of Aeromonas hydrophila was detected in very small quantities and at a few of the sampling points only.Analyses of Dissolved Organic Carbon (on average 0.3 mgC/l) and Biodegradable Dissolved Organic Carbon (less than 0.1 mgC/l, by the minimum detection threshold method) reveal a very low concentration in organics and a total absence of BDOC on all sampling points, thus confirming the good quality of the water supplied and the tact that these is very little bacteria) remise and contamination while the water is in transit.Examination of the sediments in water mains and at the bottom of reservoirs did not reveal the existence of higher forma of microorganisms.This complete diagnosis of the mirobiological and health condition of the Nice water system has brought confirmation of the good quality of the water supplied throughout its transit in the water mains. The lack of chlorination dues not cause a deterioration of the bacteriological quality of the water, the sustainable quality of which is essentially linked with the absence of BDOC which restricting the growth of bacteria.The importance of these findings on such a distinctive network as that of the Nice utility will enable this study to be used as reference for future research on the means of maintaining the quality of water in supply networks without using chlorine

No association between islet cell antibodies and coxsackie B, mumps, rubella and cytomegalovirus antibodies in non-diabetic individuals aged 7–19 years

Viral antibodies were tested in a cohort of 44 isletcell antibody-positive individuals age 7–19 years, and 44 of their islet cell antibody-negative age and sex-matched classmates selected from a population study of 4208 pupils who had been screened for islet cell antibodies. Anti-coxsackie B1-5 IgM responses were detected in 14 of 44 (32%) of the islet cell antibody-positive subjects and in 7 of 44 (16%) control subjects. This difference did not reach the level of statistical significance. None of the islet cell antibody-positive subjects had specific IgM antibodies to mumps, rubella, or cytomegalovirus. There was also no increase in the prevalence or the mean titres of anti-mumps-IgG or IgA and anti-cytomegalovirus-IgG in islet cell antibody-positive subjects compared to control subjects. These results do not suggest any association between islet cell antibodies, and possibly insulitis, with recent mumps, rubella or cytomegalo virus infection. Further studies are required to clarify the relationship between islet cell antibodies and coxsackie B virus infections

NK Cells Are Not Required for Spontaneous Autoimmune Diabetes in NOD Mice

NK cells have been shown to either promote or protect from autoimmune diseases. Several studies have examined the role of receptors preferentially expressed by NK cells in the spontaneous disease of NOD mice or the direct role of NK cells in acute induced disease models of diabetes. Yet, the role of NK cells in spontaneous diabetes has not been directly addressed. Here, we used the NOD.NK1.1 congenic mouse model to examine the role of NK cells in spontaneous diabetes. Significant numbers of NK cells were only seen in the pancreas of mice with disease. Pancreatic NK cells displayed an activated surface phenotype and proliferated more than NK cells from other tissues in the diseased mice. Nonetheless, depletion of NK cells had no effect on dendritic cell maturation or T cell proliferation. In spontaneous disease, the deletion of NK cells had no significant impact on disease onset. NK cells were also not required to promote disease induced by adoptively transferred pathogenic CD4+ T cells. Thus, NK cells are not required for spontaneous autoimmune diabetes in NOD mice

Human Ovarian Tumor Cells Escape γδ T Cell Recognition Partly by Down Regulating Surface Expression of MICA and Limiting Cell Cycle Related Molecules

Background: Mechanisms of human Vc2Vd2 T cell-mediated tumor immunity have yet to be fully elucidated. Methods and Findings: At least some tumor cell recognition is mediated by NKG2D-MICA interactions. Herein, by using MTT assay and PI-BrdU co-staining and Western-blot, we show that these Vc2Vd2 T cells can limit the proliferation of ovarian tumor cells by down regulation of apoptosis and cell cycle related molecules in tumor cells. Cell-to-cell contact is critical. cd T cell-resistant, but not susceptible ovarian tumor cells escape cd T cell-mediated immune recognition by up-regulating pErk1/2, thereby decreasing surface MICA levels. Erk1/2 inhibitor pretreatment or incubation prevents this MICA decrease, while up-regulating key cell cycle related molecules such as CDK2, CDK4 and Cyclin D1, as well as apoptosis related molecules making resistant tumor cells now vulnerable to cd T cell-mediated lysis. Conclusion: These findings demonstrate novel effects of cdT cells on ovarian tumor cells

Thermal- and Oxidative Stress Causes Enhanced Release of NKG2D Ligand-Bearing Immunosuppressive Exosomes in Leukemia/Lymphoma T and B Cells

Immune evasion from NK surveillance related to inadequate NK-cell function has been suggested as an explanation of the high incidence of relapse and fatal outcome of many blood malignancies. In this report we have used Jurkat and Raji cell lines as a model for studies of the NKG2D receptor-ligand system in T-and B cell leukemia/lymphoma. Using real-time quantitative RT-PCR and immunoflow cytometry we show that Jurkat and Raji cells constitutively express mRNA and protein for the stress-inducible NKG2D ligands MICA/B and ULBP1 and 2, and up-regulate the expression in a cell-line specific and stress-specific manner. Furthermore, we revealed by electron microscopy, immunoflow cytometry and western blot that these ligands were expressed and secreted on exosomes, nanometer-sized microvesicles of endosomal origin. Acting as a decoy, the NKG2D ligand-bearing exosomes downregulate the in vitro NKG2D receptor-mediated cytotoxicity and thus impair NK-cell function. Interestingly, thermal and oxidative stress enhanced the exosome secretion generating more soluble NKG2D ligands that aggravated the impairment of the cytotoxic response. Taken together, our results might partly explain the clinically observed NK-cell dysfunction in patients suffering from leukemia/lymphoma. The adverse effect of thermal and oxidative stress, enhancing the release of immunosuppressive exosomes, should be considered when cytostatic and hyperthermal anti-cancer therapies are designed

Regulation of immune cell function and differentiation by the NKG2D receptor

NKG2D is one of the most intensively studied immune receptors of the past decade. Its unique binding and signaling properties, expression pattern, and functions have been attracting much interest within the field due to its potent antiviral and anti-tumor properties. As an activating receptor, NKG2D is expressed on cells of the innate and adaptive immune system. It recognizes stress-induced MHC class I-like ligands and acts as a molecular sensor for cells jeopardized by viral infections or DNA damage. Although the activating functions of NKG2D have been well documented, recent analysis of NKG2D-deficient mice suggests that this receptor may have a regulatory role during NK cell development. In this review, we will revisit known aspects of NKG2D functions and present new insights in the proposed influence of this molecule on hematopoietic differentiation