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Mycoplasmas (a generic name for Mollicutes) are a predominant bacterial contaminant of cell culture and cell derived products including viruses. This prokaryote class is characterized by very small size and lack of a cell wall. Consequently, mycoplasmas escape ultrafiltration and visualization under routine microscopic examination, hence the ease with which cells in culture can be contaminated, with routinely more than 10% of cell lines being contaminated. Mycoplasma are a formidable threat both in fundamental research by perverting a whole range of cell properties and functions and in the pharmacological use of cells and cell derived products. Although many methods have been developed, there is still a need for a sensitive, universal assay. Here is reported the development and validation of a quantitative polymerase chain reaction (qPCR) based on the amplification of a 1.5 kb fragment covering the 16S rDNA of the Mollicute class by real-time PCR using universal U1 and U8 degenerate primers. The method includes the addition of a DNA loading probe to each sample to monitor DNA extraction and the absence of PCR inhibitors in the extracted DNA, a positive mycoplasma 16S rDNA traceable reference sample to exclude any accidental contamination of an unknown sample with this reference DNA, an analysis procedure based on the examination of the melting curve and the size of the PCR amplicon, followed by quantification of the number of 16S rDNA copies (with a lower limit of 19 copies) when relevant, and, if useful, the identification of the contaminating prokaryote by sequencing. The method was validated on a collection of mycoplasma strains and by testing over 100 samples of unknown contamination status including stocks of viruses requiring biosafety level 2, 3 or 4 containments. When compared to four established methods, the m16S_qPCR technique exhibits the highest sensitivity in detecting mycoplasma contamination.

Immune tolerance to self-antigens is a complex process that utilizes multiple mechanisms working in concert to maintain homeostasis and prevent autoimmunity. Considerable progress in deciphering the mechanisms controlling the activation or deletion of T cells has been made by using T cell receptor (TCR) transgenic mice. One such model is the F5 model in which CD8 T cells express a TCR specific for an epitope derived from the influenza NP68 protein. Our aim was to create transgenic mouse models expressing constitutively the NP68 epitope fused to enhanced green fluorescent protein (EGFP) in order to assess unambiguously the relative levels of NP68 epitope expressed by single cells. We used a lentiviral-based approach to generate two independent transgenic mouse strains expressing the fusion protein EGFP-NP68 under the control of CAG (CMV immediate early enhancer and the chicken β-actin promoter) or spleen focus-forming virus (SFFV) promoters. Analysis of the pattern of EGFP expression in the hematopoietic compartment showed that CAG and SFFV promoters are differentially regulated during T cell development. However, both promoters drove high EGFP-NP68 expression in dendritic cells (pDCs, CD8α(+) cDCs, and CD8α(-) cDCs) from spleen or generated in vitro following differentiation from bone-marrow progenitors. NP68 epitope was properly processed and successfully presented by dendritic cells (DCs) by direct presentation and cross-presentation to F5 CD8 T cells. The models presented here are valuable tools to investigate the priming of F5 CD8 T cells by different subsets of DCs., (Copyright © 2013 Wiley Periodicals, Inc.)

Background: The ubiquitous transcription factor Sp1 regulates the expression of a vast number of genes involved in many cellular functions ranging from differentiation to proliferation and apoptosis. Sp1 expression levels show a dramatic increase during transformation and this could play a critical role for tumour development or maintenance. Although Sp1 deregulation might be beneficial for tumour cells, its overexpression induces apoptosis of untransformed cells. Here we further characterised the functional and transcriptional responses of untransformed cells following Sp1 overexpression., Methodology and Principal Findings: We made use of wild-type and DNA-binding-deficient Sp1 to demonstrate that the induction of apoptosis by Sp1 is dependent on its capacity to bind DNA. Genome-wide expression profiling identified genes involved in cancer, cell death and cell cycle as being enriched among differentially expressed genes following Sp1 overexpression. In silico search to determine the presence of Sp1 binding sites in the promoter region of modulated genes was conducted. Genes that contained Sp1 binding sites in their promoters were enriched among down-regulated genes. The endogenous sp1 gene is one of the most down-regulated suggesting a negative feedback loop induced by overexpressed Sp1. In contrast, genes containing Sp1 binding sites in their promoters were not enriched among up-regulated genes. These results suggest that the transcriptional response involves both direct Sp1-driven transcription and indirect mechanisms. Finally, we show that Sp1 overexpression led to a modified expression of G1/S transition regulatory genes such as the down-regulation of cyclin D2 and the up-regulation of cyclin G2 and cdkn2c/p18 expression. The biological significance of these modifications was confirmed by showing that the cells accumulated in the G1 phase of the cell cycle before the onset of apoptosis., Conclusion: This study shows that the binding to DNA of overexpressed Sp1 induces an inhibition of cell cycle progression that precedes apoptosis and a transcriptional response targeting genes containing Sp1 binding sites in their promoter or not suggesting both direct Sp1-driven transcription and indirect mechanisms.

Morbillivirus infections have been known for a long time to be associated with an acute immunosuppression in their natural hosts. Here, we show that recombinant Morbillivirus nucleoproteins from canine distemper virus, peste-des-petits-ruminants virus, and Rinderpest virus bind B-lymphocytes from dogs, goats, and cattle, respectively, similarly to measles virus nucleoprotein in humans. The use of surface plasmon resonance imaging allowed the real time detection of differential interactions between Morbillivirus nucleoproteins and FcgammaRIIb (CD32). Moreover, those nucleoproteins which bind murine Fcgamma receptor inhibited the inflammatory immune responses in mice in a Fc receptor- dependent manner. In contrast, nucleoprotein from closely related Henipavirus genus, belonging to the Paramyxoviridae family as Morbillivirus, was devoid of capacity either to bind FcgammaRIIb or to inhibit inflammatory response. Altogether, these results suggest that nucleoprotein-FcR interaction is a common mechanism used by different Morbilliviruses to modulate the immune response.

Real-time PCR has been developed to genotype measles virus (MV) isolates. MV strains circulating in epidemics in Gabon in 1984, Cameroon in 2001, Morocco in 2003, and France in 2004 were investigated. We developed a real-time amplification refractory mutation system PCR (RT-AMRS PCR) using SYBR green fluorescent dye. Six pairs of primers for RT-ARMS PCR were designed to specifically amplify genotypes A, B2, B3.1, B3.2, C2, and D7. Genotypes could be differentiated by melting curve analysis. All strains were also confirmed by direct sequencing. Using the result obtained by direct sequencing and phylogenetic analysis as the reference, the accuracy of MV by RT-ARMS PCR and melting curve analysis was 97%. However, the latter method is more rapid and sensitive than the former method. This method could be a useful tool for molecular epidemiological studies of MV, providing an efficient alternative for large-scale studies.

A predominantly pig-to-human zoonotic infection caused by the novel Nipah virus emerged recently to cause severe morbidity and mortality in both animals and man. Human autopsy studies showed the pathogenesis to be related to systemic vasculitis that led to widespread thrombotic occlusion and microinfarction in most major organs especially in the central nervous system. There was also evidence of extravascular parenchymal infection, particularly near damaged vessels (Wong KT, Shieh WJ, Kumar S, Norain K, Abdullah W, Guarner J, Goldsmith CS, Chua KB, Lam SK, Tan CT, Goh KJ, Chong HT, Jusoh R, Rollin PE, Ksiazek TG, Zaki SR, Nipah Virus Pathology Working Group: Nipah virus infection: Pathology and pathogenesis of an emerging paramyxoviral zoonosis. Am J Pathol 2002, 161:2153-2167). We describe here a golden hamster (Mesocricetus auratus) model that appears to reproduce the pathology and pathogenesis of acute human Nipah infection. Hamsters infected by intranasal or intraperitoneal routes died within 9 to 29 days or 5 to 9 days, respectively. Pathological lesions were most severe and extensive in the hamster brain. Vasculitis, thrombosis, and more rarely, multinucleated endothelial syncytia, were found in blood vessels of multiple organs. Viral antigen and RNA were localized in both vascular and extravascular tissues including neurons, lung, kidney, and spleen, as demonstrated by immunohistochemistry and in situ hybridization, respectively. Paramyxoviral-type nucleocapsids were identified in neurons and in vessel walls. At the terminal stage of infection, virus and/or viral RNA could be recovered from most solid organs and urine, but not from serum. The golden hamster is proposed as a suitable model for further studies including pathogenesis studies, anti-viral drug testing, and vaccine development against acute Nipah infection.

Baf-3 cells are dependent on interleukin-3 (IL-3) for their survival and proliferation in culture. To identify anti-apoptotic pathways, we performed a retroviral-insertion mutagenesis on Baf-3 cells and selected mutants that have acquired a long term survival capacity. The phenotype of one mutant, which does not overexpress bcl-x and proliferates in the absence of IL-3, is described. We show that, in this mutant, Akt is constitutively activated leading to FKHRL1 phosphorylation and constitutive glycolytic activity. This pathway is necessary for the mutant to survive following IL-3 starvation but is not sufficient or necessary to protect cells from DNA damage-induced cell death. Indeed, inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in Baf-3 cells does not prevent the ability of IL-3 to protect cells against gamma-irradiation-induced DNA damage. This protective effect of IL-3 rather correlates with the expression of the anti-apoptotic Bcl-x protein. Taken together, these data demonstrate that the PI3K/Akt pathway is sufficient to protect cells from growth factor starvation-induced apoptosis but is not required for IL-3 inhibition of DNA damage-induced cell death.

In Baf-3 cells, IL-3 and IGF-1 both inhibit cell death. These growth factors act at least on two different pathways involved in the inhibition of apoptosis. They both upregulate Bcl-X at the mRNA and protein levels and also activate a pathway which inhibits apoptosis in the absence of protein synthesis. Recently, these two growth factors have been shown to activate the PI3-kinase-AKT pathway which leads to the phosphorylation of the pro-apoptotic Bcl-XL regulator Bad. In this study, we have investigated the role of PI3-kinase in the regulation of Bcl-X expression and in the survival of Baf-3 cells. We show that PI3-kinase activation is involved in the upregulation of Bcl-X mRNA induced by both IL-3 and IGF-1. Moreover, PI3-kinase activity is also necessary for inhibition of apoptosis and caspase regulation by IGF-1 but not IL-3.