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  • 1
    Keywords: RECEPTOR ; CELLS ; EXPRESSION ; IN-VITRO ; CELL ; SYSTEM ; TOOL ; GENE ; cell line ; TRANSDUCTION ; INFECTION ; murine ; recombination ; antibodies ; antibody ; TARGET ; virus ; MOUSE ; hormone ; VECTORS ; CELL-LINE ; FUSION ; LINE ; CARCINOMA-CELLS ; MAMMALIAN-CELLS ; HEMATOPOIETIC PROGENITOR CELLS ; RETROVIRAL VECTORS ; VIRAL VECTORS ; PLASMID DNA ; TRANSGENE EXPRESSION ; HIGH-TITER ; gene transfer,ES cells,MESV retroviral vectors,adenoviral vectors,Cre recombinase,Cre.PR fusion ; RECOMBINANT ADENOVIRUS ; SOMATIC MUTAGENESIS
    Abstract: Background Genetic modification of embryonic stem (ES) cells represents a powerful tool for transgenic and developmental experiments. We report that retroviral constructs based on murine embryonal stem cell virus (MESV) can efficiently deliver and express Cre recombinase or a post-translationally inducible Cre-Progesterone receptor (Cre.PR) fusion in mouse fibroblasts and ES cells.Methods To study the vectors a sensitive reporter cell line, 3TZ, was derived from the murine 3T6 fibroblast line that expresses beta-galactosidase only upon Cre-mediated recombination. This was used together with the ROSA26-R ES cell Cre-reporter system or unmodified mouse ES cells as targets of infection. Efficiency of gene transfer was evaluated immunohistochemically by the use of an anti-Cre polyclonal antibody, and by monitoring the expression of beta-galactosidase.Results Infection of the 3TZ cells with high titer 718C or 719CP virus revealed efficient gene transduction of constitutive or hormone-inducible recombinase activity, respectively. The vectors efficiently transduced murine ES cells with Cre, Cre-PR (fusion of Cre and progesterone receptor) or beta-galactosidase. Cre-mediated recombination in more than 60% of ROSA26-R ES cells was achieved when infected by a VSV-G-pseudotyped MESV retrovirus at MOI of 50.Conclusions The MESV-based retroviral systems, when combined with hormone inducible Cre, represent efficient tools for the transfer of Cre activity in ES cells. Copyright (C) 2004 John Wiley Sons, Ltd
    Type of Publication: Journal article published
    PubMed ID: 14716675
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  • 2
    Keywords: EXPRESSION ; PROTEIN ; INFECTION ; CERVICAL-CANCER ; HUMAN KERATINOCYTES ; EPITHELIAL-CELLS ; DEGRADATION ; NECK-CANCER ; AUTOPHAGY ; INFLAMMASOMES
    Abstract: Infections with high-risk human papillomaviruses (HPVs) are causally involved in the development of anogenital cancer. HPVs apparently evade the innate immune response of their host cells by dysregulating immunomodulatory factors such as cytokines and chemokines, thereby creating a microenvironment that favors malignancy. One central key player in the immune surveillance interactome is interleukin-1 beta (IL-1beta) which not only mediates inflammation, but also links innate and adaptive immunity. Because of its pleiotropic physiological effects, IL-1beta production is tightly controlled on transcriptional, post-translational and secretory levels. Here, we describe a novel mechanism how the high-risk HPV16 E6 oncoprotein abrogates IL-1beta processing and secretion in a NALP3 inflammasome-independent manner. We analyzed IL-1beta regulation in immortalized keratinocytes that harbor the HPV16 E6 and/or E7 oncogenes as well as HPV-positive cervical tumor cells. While in primary and in E7-immortalized human keratinocytes the secretion of IL-1beta was highly inducible upon inflammasome activation, E6-positive cells did not respond. Western blot analyses revealed a strong reduction of basal intracellular levels of pro-IL-1beta that was independent of dysregulation of the NALP3 inflammasome, autophagy or lysosomal activity. Instead, we demonstrate that pro-IL-1beta is degraded in a proteasome-dependent manner in E6-positive cells which is mediated via the ubiquitin ligase E6-AP and p53. Conversely, in E6- and E6/E7-immortalized cells pro-IL-1beta levels were restored by siRNA knock-down of E6-AP and simultaneous recovery of functional p53. In the context of HPV-induced carcinogenesis, these data suggest a novel post-translational mechanism of pro-IL-1beta regulation which ultimately inhibits the secretion of IL-1beta in virus-infected keratinocytes. The clinical relevance of our results was further confirmed in HPV-positive tissue samples, where a gradual decrease of IL-1beta towards cervical cancer could be discerned. Hence, attenuation of IL-1beta by the HPV16 E6 oncoprotein in immortalized cells is apparently a crucial step in viral immune evasion and initiation of malignancy.
    Type of Publication: Journal article published
    PubMed ID: 23935506
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  • 3
    Keywords: CANCER ; PROTECTION ; INFECTION ; antibodies ; LESIONS ; TYPE-16 ; VACCINES ; VIRUS-LIKE PARTICLES ; HPV16 ; Rabbits
    Abstract: The amino terminus of the human papillomavirus minor capsid protein L2 contains a major cross-neutralizing epitope that provides the basis for the development of a broadly protective HPV vaccine. This attainable broad protection would eliminate one of the major drawbacks of the commercial L1-based prophylactic vaccines. In this study, we asked whether there are natural variants of the L2 cross-neutralizing epitope and if these variants provide means for immune escape from vaccine-induced anti-L2 antibodies. For this, we isolated in silico and in vitro, a total of 477 L2 sequences of HPV types 16, 18, 31, 45, 51, 52 and 58. We identified natural L2 epitope variants for HPV 18, 31, 45 and 51. To determine whether these variants escape L2-directed neutralization, we generated pseudovirions encompassing the natural variants and tested these in an in vitro neutralization assay using monoclonal and polyclonal antibodies. Our results indicate that natural variants of the L2 major neutralizing epitope are frequent among two different study populations from Germany and Mongolia and in the GenBank database. Of two identified HPV 31 L2 single amino acid variants, one could be neutralized well, while the other variant was neutralized very poorly. We also observed that single amino acid variants of HPV 18 and 45 are neutralized well while a HPV 18 double variant was neutralized at significantly lower rates, indicating that L2 variants have to be accounted for when developing HPV L2-based prophylactic vaccines.
    Type of Publication: Journal article published
    PubMed ID: 22961598
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