Identification of Naturally Processed Hepatitis C Virus-Derived Major Histocompatibility Complex Class I Ligands

Fine mapping of human cytotoxic T lymphocyte (CTL) responses against hepatitis C virus (HCV) is based on external loading of target cells with synthetic peptides which are either derived from prediction algorithms or from overlapping peptide libraries. These strategies do not address putative host and viral mechanisms which may alter processing as well as presentation of CTL epitopes. Therefore, the aim of this proof-of-concept study was to identify naturally processed HCV-derived major histocompatibility complex (MHC) class I ligands. To this end, continuous human cell lines were engineered to inducibly express HCV proteins and to constitutively express high levels of functional HLA-A2. These cell lines were recognized in an HLA-A2-restricted manner by HCV-specific CTLs. Ligands eluted from HLA-A2 molecules isolated from large-scale cultures of these cell lines were separated by high performance liquid chromatography and further analyzed by electrospray ionization quadrupole time of flight mass spectrometry (MS)/tandem MS. These analyses allowed the identification of two HLA-A2-restricted epitopes derived from HCV nonstructural proteins (NS) 3 and 5B (NS31406–1415 and NS5B2594–2602). In conclusion, we describe a general strategy that may be useful to investigate HCV pathogenesis and may contribute to the development of preventive and therapeutic vaccines in the future

Bewegtbildstrategien von Tageszeitungen in Baden-Württemberg

Der Fokus dieser Arbeit liegt auf den Bewegtbildstrategien von Tageszeitungen in Baden-Württemberg. Es sollte aufgezeigt werden welche strategischen Vorgehensweisen die Lokalzeitungen in Baden-Württemberg verwenden und wie sie diese Umsetzen. Weiter sollten Faktoren extrahiert werden, die als erfolgskritisch angenommen werden können. Herausgefunden wurde, dass alle befragten Zeitungsverlage eine konvergenzinduzierte Cross-Media-Strategie verfolgen, in der die Diversifikation vertikal verläuft. Das heißt die Zeitungen sind meist selbst Produzent des Bewegtbilds und nutzen die wechselseitigen Synergieeffekte der redaktionellen Kompetenzen und multimedialen Distributionskanäle. Weiter kann festgestellt werden, dass Im Bereich Bewegtbild Ansätze einer Nischen- oder Fokusstrategie zu erkennen sind. Als Erfolgsfaktoren können vor allem die lokale Kompetenz, die Format- und Themenwahl von Videos sowie die Redaktionsorganisation, die stark mit der Nutzung von Synergieeffekten zusammenhängt, angenommen werden.The focus of this paper is on the video strategies of daily newspapers in Baden-Württemberg. The aim was to show which strategic approaches local newspapers in Baden-Württemberg use and how they implement them. Furthermore, factors should be extracted that can be assumed to be critical for success. It was found that all the newspaper publishers surveyed pursue a convergence-induced cross-media strategy in which diversification is vertical. This means that the newspapers are mostly producers of the moving image themselves and use the mutual synergy effects of editorial competencies and multimedia distribution channels. It can also be seen that there are signs of a niche or focus strategy on video. Local competence, the choice of the video topic, and editorial organization, which is strongly related to the use of synergy effects, can be assumed to be the main success factors

A Dynamic View of Hepatitis C Virus Replication Complexes▿ ‡

Hepatitis C virus (HCV) replicates its genome in a membrane-associated replication complex (RC). Specific membrane alterations, designated membranous webs, represent predominant sites of HCV RNA replication. The principles governing HCV RC and membranous web formation are poorly understood. Here, we used replicons harboring a green fluorescent protein (GFP) insertion in nonstructural protein 5A (NS5A) to study HCV RCs in live cells. Two distinct patterns of NS5A-GFP were observed. (i) Large structures, representing membranous webs, showed restricted motility, were stable over many hours, were partitioned among daughter cells during cell division, and displayed a static internal architecture without detectable exchange of NS5A-GFP. (ii) In contrast, small structures, presumably representing small RCs, showed fast, saltatory movements over long distances. Both populations were associated with endoplasmic reticulum (ER) tubules, but only small RCs showed ER-independent, microtubule (MT)-dependent transport. We suggest that this MT-dependent transport sustains two distinct RC populations, which are both required during the HCV life cycle

Generation and Preclinical Characterization of a Fc-optimized GITR-Ig Fusion Protein for Induction of NK Cell Reactivity Against Leukemia

Natural killer (NK) cells are cytotoxic lymphocytes that largely contribute to the efficacy of therapeutic strategies like allogenic stem cell transplantation in acute myeloid leukemia (AML) and application of Rituximab in chronic lymphocytic leukemia (CLL). The tumor necrosis factor (TNF) family member GITR ligand (GITRL) is frequently expressed on leukemia cells in AML and CLL and impairs the reactivity of NK cells which express GITR and upregulate its expression following activation. We developed a strategy to reinforce NK anti-leukemia reactivity by combining disruption of GITR–GITRL interaction with targeting leukemia cells for NK antibody-dependent cellular cytotoxicity (ADCC) using GITR-Ig fusion proteins with modified Fc moieties. Neutralization of leukemia-expressed GITRL by the GITR domain enhanced cytotoxicity and cytokine production of NK cells depending on activation state with NK reactivity being further largely dependent on the engineered affinity of the fusion proteins to the Fc receptor. Compared with wild-type GITR-Ig, treatment of primary AML and CLL cells with mutants containing a S239D/I332E modification potently increased cytotoxicity, degranulation, and cytokine production of NK cells in a target-antigen–dependent manner with additive effects being observed with CLL cells upon parallel exposure to Rituximab. Fc-optimized GITR-Ig may thus constitute an attractive means for immunotherapy of leukemia that warrants clinical evaluation

Identification of naturally processed HLA-A2 ligands.

<p>Following large-scale expansion, cell pellets were lysed and MHC class I molecules with bound ligands were purified by immunoprecipitation. Subsequently, ligands were eluted and separated by high performance liquid chromatography (HPLC). Fractions of interest were sequenced by mass spectrometry.</p

Detection and characterization of peptide NS3_1406–1415 in MHC class I ligands isolated from UNS3-4A/A2-27.35 cells.

<p>Naturally processed MHC class I ligands of UNS3-4A/A2-27.35 cells were separated by high performance liquid chromatography (HPLC) and analyzed by mass spectrometry. (A) A chromatogram of the total ion current (TIC) is shown. (B) The mass chromatogram of ionized peptides with m/z = 499.3 reveals a peak at retention time t = 49.5 min that correlates with (C) a signal of the synthetic peptide NS3_1406–1415 (KLVALGINAV) which elutes after 51.3 min. In all graphs, HPLC retention times are shown on the abscissa and relative signal intensity on the ordinate. Small insets in panels B and C show mass spectra of peptides eluted at the indicated time points. (D) MSMS spectrum of the synthetic peptide NS3_1406–1415 (KLVALGINAV). (E) MSMS spectrum of the natural peptide isolated from UNS3-4A/A2-27.35 cells, revealing amino acid sequence KLVALGINAV identical to the synthetic peptide shown in panel A. Identified amino acid sequences of peptide fragments are indicated on the top of the peaks. Due to the protonated N-terminal lysine residue, the b series of peptide fragments is dominating the MSMS spectra.</p

Cell lines UNS3-4A/A2-27.35 and UHCV/A2-27.

<p>(A) Indirect immunofluorescence microscopy of UNS3-4A/A2-27.35 and UHCV/A2-27 cells cultured for 48 h in the presence (+ tet) or absence (− tet) of tetracycline. Monoclonal antibody 1B6 against HCV NS3 was used as primary antibody. (B) Immunoblot analysis of UNS3-4A/A2-27.35 and UHCV/A2-27 cells were cultured for 48 h in the presence (+ tet) or absence (− tet) of tetracycline. Monoclonal antibodies 1B6 against HCV NS3 or 11H against NS5A were used as primary antibodies. (C) Left panel: HLA-A2 surface expression of UNS3-4A/A2-27.35 (blue histogram) compared to the founder cell line UNS3-4A-24 (red histogram). Right panel: HLA-A2 surface expression of UHCV/A2-27 (blue histogram) compared to the founder cell line UHCVcon-57.3 (red histogram).</p

Erratum to: Azimuthal asymmetries of charged hadrons produced in high-energy muon scattering off longitudinally polarised deuterons

The original version of this article unfortunately contained a mistake