Activation of interferon regulatory factor 3 by replication-competent Vaccinia viruses improves antitumor efficacy mediated by T-cell responses

Recently, oncolytic vaccinia viruses (VACVs) have shown their potential to provide for clinically effective cancer treatments. The reason for this clinical usefulness is not only the direct destruction of infected cancer cells but also activation of immune responses directed against tumor antigens. For eliciting a robust antitumor immunity, a dominant T helper 1 (Th1) cell differentiation of the response is preferred, and such polarization can be achieved by activating the Toll-like receptor 3 (TLR3)-interferon regulatory factor 3 (IRF3) signaling pathway. However, current VACVs used as oncolytic viruses to date still encode several immune evasion proteins involved in the inhibition of this signaling pathway. By inactivating genes of selected regulatory virus proteins, we aimed for a candidate virus with increased potency to activate cellular antitumor immunity but at the same time with a fully maintained replicative capacity in cancer cells. The removal of up to three key genes (C10L, N2L, and C6L) from VACV did not reduce the strength of viral replication, both in vitro and in vivo, but resulted in the rescue of IRF3 phosphorylation upon infection of cancer cells. In syngeneic mouse tumor models, this activation translated to enhanced cytotoxic T lymphocyte (CTL) responses directed against tumor-associated antigens and neo-epitopes and improved antitumor activity

Combination of a fast white light interferometer with a phase-shifting interferometric line sensor for form measurements of precision components

Modern industrial fabrication processes put high requirements on the quality of the surface form of precision-machined components, e.g. optical lenses or microelectromechanical systems (MEMS). Optical sensors provide high precision non-contact surface measurement to verify these quality requirements, even on fragile surfaces. The low-cost line-scanning interferometer that is presented in this contribution is based on a Michelson interferometer configuration in combination with a high-speed line-scan camera. The sensor can operate in scanning white-light or in optical path length modulation (OPLM) mode. The white-light mode is used to automatically align the sensor perpendicular in the working distance of 13 mm to the surface of the specimen. In OPLM-mode, an oscillating reference mirror and a band-pass filtered light source are used, to measure the form of a radial symmetric specimen with a diameter of up to 25.4 mm with interferometric accuracy. Several overlapping ring-shaped sub-apertures are measured iteratively in different radial positions until the whole surface is scanned. The sub-apertures are stitched together to reconstruct the complete 3D-topography, while overlapping areas can be used to correct movement errors of the scanning axes. This concept is highly adaptive and can be applied to many different specimen geometries e.g. planes, spheres or aspheric glass lenses

On the Enigmatic Hook of the Metaradiophryids (Alveolata, Ciliophora)

The astomatous metaradiophryids are ciliates which live endosymbiotically in earthworms (Annelida, Lumbricidae). Their prominent hook apparatus is demonstrated in detail in light micrographs of living organisms as well as in scanning electron micrographs of Parducz-fixed cells. Since it was first observed, this structure has been interpreted as a ‘holdfast’ organelle preventing ciliates from being expelled prematurely from the intestine of the worm along with its excrements. No active movement of the hook has been reported in earlier papers or in our recent studies. Nevertheless, a detailed description of different parts of the hook apparatus exists in older literature, including a hypothesis on how these elements interact with each other to function as a holdfast device – without any experimental evidence. The suspected mode of function of this structure is questioned and critically discussed

Critical Role of Perforin-dependent CD8+ T Cell Immunity for Rapid Protective Vaccination in a Murine Model for Human Smallpox

Vaccination is highly effective in preventing various infectious diseases, whereas the constant threat of new emerging pathogens necessitates the development of innovative vaccination principles that also confer rapid protection in a case of emergency. Although increasing evidence points to T cell immunity playing a critical role in vaccination against viral diseases, vaccine efficacy is mostly associated with the induction of antibody responses. Here we analyze the immunological mechanism(s) of rapidly protective vaccinia virus immunization using mousepox as surrogate model for human smallpox. We found that fast protection against lethal systemic poxvirus disease solely depended on CD4 and CD8 T cell responses induced by vaccination with highly attenuated modified vaccinia virus Ankara (MVA) or conventional vaccinia virus. Of note, CD4 T cells were critically required to allow for MVA induced CD8 T cell expansion and perforin-mediated cytotoxicity was a key mechanism of MVA induced protection. In contrast, selected components of the innate immune system and B cell-mediated responses were fully dispensable for prevention of fatal disease by immunization given two days before challenge. In conclusion, our data clearly demonstrate that perforin-dependent CD8 T cell immunity plays a key role in MVA conferred short term protection against lethal mousepox. Rapid induction of T cell immunity might serve as a new paradigm for treatments that need to fit into a scenario of protective emergency vaccination

Automated evaluation of autoantibodies on human epithelial-2 cells as an approach to standardize cell-based immunofluorescence tests

INTRODUCTION: Analysis of autoantibodies (AAB) by indirect immunofluorescence (IIF) is a basic tool for the serological diagnosis of systemic rheumatic disorders. Automation of autoantibody IIF reading including pattern recognition may improve intra- and inter-laboratory variability and meet the demand for cost-effective assessment of large numbers of samples. Comparing automated and visual interpretation, the usefulness for routine laboratory diagnostics was investigated. METHODS: Autoantibody detection by IIF on human epithelial-2 (HEp-2) cells was conducted in a total of 1222 consecutive sera of patients with suspected systemic rheumatic diseases from a university routine laboratory (n = 924) and a private referral laboratory (n = 298). IIF results from routine diagnostics were compared with a novel automated interpretation system. RESULTS: Both diagnostic procedures showed a very good agreement in detecting AAB (kappa = 0.828) and differentiating respective immunofluorescence patterns. Only 98 (8.0%) of 1222 sera demonstrated discrepant results in the differentiation of positive from negative samples. The contingency coefficients of chi-square statistics were 0.646 for the university laboratory cohort with an agreement of 93.0% and 0.695 for the private laboratory cohort with an agreement of 90.6%, P < 0.0001, respectively. Comparing immunofluorescence patterns, 111 (15.3%) sera yielded differing results. CONCLUSIONS: Automated assessment of AAB by IIF on HEp-2 cells using an automated interpretation system is a reliable and robust method for positive/negative differentiation. Employing novel mathematical algorithms, automated interpretation provides reproducible detection of specific immunofluorescence patterns on HEp-2 cells. Automated interpretation can reduce drawbacks of IIF for AAB detection in routine diagnostics providing more reliable data for clinicians

Identifying eastern Baltic cod nursery grounds using hydrodynamic modelling: knowledge for the design of Marine Protected areas

Knowledge of the spatial and temporal distribution of juvenile cod is essential to closing the life cycle in population dynamic models, and it is a prerequisite for the design of Marine Protected Areas (MPAs) aiming at the protection of juveniles. In this study, we use a hydrodynamic model to examine the spatial distribution of eastern Baltic cod larvae and early juveniles. The transport patterns of the larvae spawned at the three major spawning grounds in the central Baltic Sea were investigated by drift model simulations for the period 1979–2004. We analysed potential habitats for their suitability for juvenile settlement, i.e. the change from pelagic to demersal life. The results revealed a clear dependence of the probability for successful settling on wind-induced drift of larval cod, which is controlled by the local atmospheric conditions over the Baltic Sea. Furthermore, we found evidence that the final destinations of juvenile cod drift routes are affected by decadal climate variability. Application of the methodology to MPA design is discussed, e.g. identifying the overlap of areas with a high probability of successful juvenile cod settlement and regions of high fishing effort in small-meshed fisheries targeting sprat and herring

Molecular structure, DNA binding mode, photophysical properties and recommendations for use of SYBR Gold

SYBR Gold is a commonly used and particularly bright fluorescent DNA stain, however, its chemical structure is unknown and its binding mode to DNA remains controversial. Here, we solve the structure of SYBR Gold by NMR and mass spectrometry to be 2-N-(3-dimethylaminopropyl)-N-propylamino]-4-2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene-1-phenyl-quinolinium and determine its extinction coefficient. We quantitate SYBR Gold binding to DNA using two complementary approaches. First, we use single-molecule magnetic tweezers (MT) to determine the effects of SYBR Gold binding on DNA length and twist. The MT assay reveals systematic lengthening and unwinding of DNA by 19.1° ± 0.7° per molecule upon binding, consistent with intercalation, similar to the related dye SYBR Green I. We complement the MT data with spectroscopic characterization of SYBR Gold. The data are well described by a global binding model for dye concentrations ≤2.5~μM, with parameters that quantitatively agree with the MT results. The fluorescence increases linearly with the number of intercalated SYBR Gold molecules up to dye concentrations of ∼2.5~μM, where quenching and inner filter effects become relevant. In summary, we provide a mechanistic understanding of DNA-SYBR Gold interactions and present practical guidelines for optimal DNA detection and quantitative DNA sensing applications using SYBR Gold