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

Weaving Nanoscale Cloth through Electrostatic Templating

Here we disclose a simple route to nanoscopic 2D woven structures reminiscent of the methods used to produce macroscopic textiles. We find that the same principles used in macroscopic weaving can be applied on the nanoscale to create two-dimensional molecular cloth from polymeric strands, a molecular thread. The molecular thread is composed of Co6Se8(PEt3)4L2 superatoms that are bridged with L = benzene bis-1,4-isonitrile to form polymer strands. As the superatoms that make up the polymer chain are electrochemically oxidized, they are electrostatically templated by a nanoscale anion, the tetragonal Lindqvist polyoxometalate Mo6O192–. The tetragonal symmetry of the dianionic template creates a nanoscale version of the box weave. The crossing points in the weave feature π-stacking of the bridging linker. By examining the steps in the weaving process with single crystal X-ray diffraction, we find that the degree of polymerization at the crossing points is crucial in the cloth formation. 2D nanoscale cloth will provide access to a new generation of smart, multifunctional materials, coatings, and surfaces

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

Beneficial autoimmunity at body surfaces – immune surveillance and rapid type 2 immunity regulate tissue homeostasis and cancer

Epithelial cells line body surface tissues and provide a physicochemical barrier to the external environment. Frequent microbial and non-microbial challenges such as those imposed by mechanical disruption, injury or exposure to noxious environmental substances including chemicals, carcinogens, ultraviolet-irradiation or toxins cause activation of epithelial cells with release of cytokines and chemokines as well as alterations in the expression of cell surface ligands. Such display of epithelial stress is rapidly sensed by tissue resident immunocytes, which can directly interact with self-moieties on epithelial cells and initiate both local and systemic immune responses. Epithelial cells are thus key drivers of immune surveillance at body surface tissues. However, epithelial cells have a propensity to drive type 2 immunity (rather than type 1) upon non-invasive challenge or stress – a type of immunity whose regulation and function still remain enigmatic. Here we review the induction and possible role of type 2 immunity in epithelial tissues and propose that rapid immune surveillance and type 2 immunity are key regulators of tissue homeostasis and carcinogenesis

The Human Herpesvirus-7 (HHV-7) U21 Immunoevasin Subverts NK-Mediated Cytoxicity through Modulation of MICA and MICB

Herpesviruses have evolved numerous immune evasion strategies to facilitate establishment of lifelong persistent infections. Many herpesviruses encode gene products devoted to preventing viral antigen presentation as a means of escaping detection by cytotoxic T lymphocytes. The human herpesvirus-7 (HHV-7) U21 gene product, for example, is an immunoevasin that binds to class I major histocompatibility complex molecules and redirects them to the lysosomal compartment. Virus infection can also induce the upregulation of surface ligands that activate NK cells. Accordingly, the herpesviruses have evolved a diverse array of mechanisms to prevent NK cell engagement of NK-activating ligands on virus-infected cells. Here we demonstrate that the HHV-7 U21 gene product interferes with NK recognition. U21 can bind to the NK activating ligand ULBP1 and reroute it to the lysosomal compartment. In addition, U21 downregulates the surface expression of the NK activating ligands MICA and MICB, resulting in a reduction in NK-mediated cytotoxicity. These results suggest that this single viral protein may interfere both with CTL-mediated recognition through the downregulation of class I MHC molecules as well as NK-mediated recognition through downregulation of NK activating ligands

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

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