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Chhatwal P.,Center for Experimental and Clinical Infection Research
PloS one

Hepatitis C virus (HCV) patients with high serum levels of bile acids (BAs) respond poorly to IFN therapy. BAs have been shown to increase RNA-replication of genotype 1 but not genotype 2a replicons. Since BAs modulate lipid metabolism including lipoprotein secretion and as HCV depends on lipids and lipoproteins during RNA-replication, virus production and cell entry, BAs may affect multiple steps of the HCV life cycle. Therefore, we analyzed the influence of BAs on individual steps of virus replication. We measured replication of subgenomic genotype (GT) 1b and 2a RNAs as well as full-length GT2a genomes in the presence of BAs using quantitative RT-PCR and luciferase assays. Cell entry was determined using HCV pseudoparticles (HCVpp). Virus assembly and release were quantified using a core-specific ELISA. Replicon chimeras were employed to characterize genotype-specific modulation of HCV by BAs. Lunet CD81/GFP-NLS-MAVS cells were used to determine infection of Con1 particles. BAs increased RNA-replication of GT1b replicons up to 10-fold but had no effect on subgenomic GT2a replicons both in Huh-7 and HuH6 cells. They did not increase viral RNA translation, virus assembly and release or cell entry. Lowering replication efficiency of GT2a replicons rendered them susceptible to stimulation by BAs. Moreover, replication of full length GT1b with or without replication enhancing mutations and GT2a genomes were also stimulated by BAs. Bile acids specifically enhance RNA-replication. This is not limited to GT1, but also holds true for GT2a full length genomes and subgenomic replicons with low replication capacity. The increase of HCV replication by BAs may influence the efficacy of antiviral treatment in vivo and may improve replication of primary HCV genomes in cell culture. Source

Bitzegeio J.,Center for Experimental and Clinical Infection Research
PLoS pathogens

Hepatitis C virus (HCV) naturally infects only humans and chimpanzees. The determinants responsible for this narrow species tropism are not well defined. Virus cell entry involves human scavenger receptor class B type I (SR-BI), CD81, claudin-1 and occludin. Among these, at least CD81 and occludin are utilized in a highly species-specific fashion, thus contributing to the narrow host range of HCV. We adapted HCV to mouse CD81 and identified three envelope glycoprotein mutations which together enhance infection of cells with mouse or other rodent receptors approximately 100-fold. These mutations enhanced interaction with human CD81 and increased exposure of the binding site for CD81 on the surface of virus particles. These changes were accompanied by augmented susceptibility of adapted HCV to neutralization by E2-specific antibodies indicative of major conformational changes of virus-resident E1/E2-complexes. Neutralization with CD81, SR-BI- and claudin-1-specific antibodies and knock down of occludin expression by siRNAs indicate that the adapted virus remains dependent on these host factors but apparently utilizes CD81, SR-BI and occludin with increased efficiency. Importantly, adapted E1/E2 complexes mediate HCV cell entry into mouse cells in the absence of human entry factors. These results further our knowledge of HCV receptor interactions and indicate that three glycoprotein mutations are sufficient to overcome the species-specific restriction of HCV cell entry into mouse cells. Moreover, these findings should contribute to the development of an immunocompetent small animal model fully permissive to HCV. Source

Bald T.,University of Bonn | Landsberg J.,University of Bonn | Lopez-Ramos D.,University of Bonn | Renn M.,University of Bonn | And 10 more authors.
Cancer Discovery

Infiltration of human melanomas with cytotoxic immune cells correlates with spontaneous type I IFN activation and a favorable prognosis. Therapeutic blockade of immune-inhibitory receptors in patients with preexisting lymphocytic infiltrates prolongs survival, but new complementary strategies are needed to activate cellular antitumor immunity in immune cell-poor melanomas. Here, we show that primary melanomas in Hgf-Cdk4R24C mice, which imitate human immune cell-poor melanomas with a poor outcome, escape IFN-induced immune surveillance and editing. Peritumoral injections of immunostimulatory RNA initiated a cytotoxic inflammatory response in the tumor microenvironment and significantly impaired tumor growth. This critically required the coordinated induction of type I IFN responses by dendritic, myeloid, natural killer, and T cells. Importantly, antibodymediated blockade of the IFN-induced immune-inhibitory interaction between PD-L1 and PD-1 receptors further prolonged the survival. These results highlight important interconnections between type I IFNs and immune-inhibitory receptors in melanoma pathogenesis, which serve as targets for combination immunotherapies. SIGNIFICANCE: Using a genetically engineered mouse melanoma model, we demonstrate that targeted activation of the type I IFN system with immunostimulatory RNA in combination with blockade of immune-inhibitory receptors is a rational strategy to expose immune cell-poor tumors to cellular immune surveillance. © 2014 American Association for Cancer Research. Source

Junge N.,Hannover Medical School | Mingozzi F.,Genethon | Ott M.,Center for Experimental and Clinical Infection Research | Baumann U.,Hannover Medical School
Journal of Pediatric Gastroenterology and Nutrition

Liver-based metabolic diseases account for a substantial burden of childhood diseases. In most patients, treatment is often limited to supportive measures and liver transplantation is ultimately required. Even despite the excellent long-term outcome of liver transplantation, the procedure is associated with a significant morbidity and mortality. Gene therapy, in contrast, has great potential to save lives, improve the quality of life, and offer few risks and adverse effects compared with present therapies including liver transplantation. The most promising results to date in liver gene transfer have been achieved with adeno-associated virus. Although safety issues, such as immunogenicity of vector and/or transgene product, remain an important concern, gene therapy is ready for clinical trials in adults and adolescents. Developing and testing safe approaches for efficient and long-term stable applications in newborns and small children, such as targeted integration and gene correction, is one of the remaining future challenges. © Copyright 2015 by ESPGHAN and NASPGHAN. Unauthorized reproduction of this article is prohibited. Source

Rath P.-M.,University of Duisburg - Essen | Schoch B.,University of Duisburg - Essen | Adamzik M.,University of Duisburg - Essen | Steinmann E.,Center for Experimental and Clinical Infection Research | And 2 more authors.

Purpose: This prospective observational cohort study assessed the use of a multiplex real-time polymerase chain reaction (PCR) assay alone and in conjunction with biomarkers for the diagnosis of ventriculostomy-related meningitis in neurosurgery intensive care unit (ICU) patients with external ventricular drainage (EVD). Methods: Concentrations of intrathecal biomarkers, including lactate and interleukin 6 (IL-6), were measured, and cerebrospinal fluid (CSF) was examined microbiologically by blood culture BACTEC bottles in 62 CSF samples from 41 patients with EVD. A portion of each sample was also tested with a commercially available PCR assay that simultaneously detects 25 species of bacteria and fungi [SeptiFast (SF)]. Receiver operating characteristic curve analysis was used to compare biomarker concentrations with SF and culture results. Results: Seventeen (27 %) samples tested positive and 40 (65 %) tested negative for pathogens by both culture and SF. One pathogen was detected only by SF. Four samples tested positive by culture but negative by SF; in 3 of these, the isolates were considered to be contaminants. In comparison to CSF culture SF showed a larger area under the curve for IL-6 (0.90; 95 % CI 0.83-0.98) versus 0.70 (95 % CI 0.46-0.80) and for lactate (0.77; 95 % CI 0.63-0.93) versus 0.65 (95 % CI 0.50-0.80). In 94 % (17/18) of positive SF samples the results were obtained on the same day whereas the overall mean of the time-to-positivity of BACTEC bottles was 21.6 h. Conclusions: The diagnosis of EVD-related ventriculo-meningitis in neurosurgical ICU patients can be established in a rapid manner using a multiplex PCR assay on CSF samples in combination with intrathecal biomarkers. © 2014 Springer-Verlag. Source

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