Cytoskeleton adaptation to stretchable surface relaxation improves adherent cryopreservation of human mesenchymal stem cells.

Adherent cell systems are usually dissociated before being cryopreserved, as standard protocols are established for cells in suspension. The application of standard procedures to more complex systems, sensitive to dissociation, such as adherent monolayers, especially comprising mature cell types, or tissues, remains unsatisfactory. Uncontrolled cell detachment due to intracellular tensile stress, membrane ruptures and damages of adhesion proteins are common during freezing and thawing of cell monolayers. However, many therapeutically relevant cell systems grow adherently to develop their native morphology and functionality, but lose their integrity after dissociation. The hypothesis is that cells on stretchable substrates have a more adaptable cytoskeleton and membrane, reducing cryopreservation-induced stress. Our studies investigate the influence of stretchable surfaces on the cryopreservation of adherent cells to avoid harmful dissociation and expedite post-thawing cultivation of functional cells. A stretching apparatus for defined radial stretching, consisting of silicone vessels and films with specific surface textures for cell culture, were developed. Adherent human umbilical cord mesenchymal stem cells (hUC-MSCs) were cultivated on a stretched silicone film within the vessel, forming a monolayer that were compressed by relaxation, while remaining attached to the relaxed film. Compressed hUC-MSCs, which were cryopreserved adherently showed higher viability and less detachment after thawing compared to control cells without compression. Within three to seven days post-thawing, the hUC-MSCs recovered, and the monolayer reformed. These experiments support the hypothesis that cryopreservation success of adherent cell systems is enhanced by improved adaptability of the cytoskeleton and cell membrane, opening up new approaches in cryobiotechnology.

Reducing electromagnetic interference in MR thermometry: A comparison of setup configurations for MR-guided microwave ablations.

Magnetic Resonance (MR) thermometry is used for the monitoring of MR-guided microwave ablations (MWA), and for the intraoperative evaluation of ablation regions. Nevertheless, the accuracy of temperature mapping may be compromised by electromagnetic interference emanating from the microwave (MW) generator. This study evaluated different setups for improving magnetic resonance imaging (MRI) during MWA with a modified MW generator. MWA was performed in 15 gel phantoms comparing three setups: The MW generator was placed outside the MR scanner room, either connected to the MW applicator using a penetration panel with a radiofrequency (RF) filter and a 7 m coaxial cable (Setup 1), or through a waveguide using a 5 m coaxial cable (Setup 2). Setup 3 employed the MW generator within the MR scan room, connected by a 5 m coaxial cable. The coaxial cables in setups 2 and 3 were modified with custom shielding to reduce interference. The setups during ablation (active setup) were compared to a reference setup without the presence of the MW system. Thermometry and thermal dose maps (CEM43 model) were compared for the three configurations. Primary endpoints for assessment were signal-to-noise ratio (SNR), temperature precision, Sørensen-Dice-Coefficient (DSC), and RF-noise spectra. Setup 3 showed highly significant electromagnetic interference during ablation with a SNR decrease by -60.4%±13.5% (p<0.001) compared to reference imaging. For setup 1 and setup 2 no significant decrease in SNR was measured with differences of -2.9%±9.8% (p=0.6) and -1.5%±12.8% (p=0.8), respectively. SNR differences were significant between active setups 1 and 3 with -51.2%±16.1% (p<0.001) and between active setups 2 and 3 with -59.0%±15.5% (p<0.001) but not significant between active setups 1 and 2 with 19.0%±13.7% (p=0.09). Furthermore, no significant differences were seen in temperature precision or DSCs between all setups, ranging from 0.33 °C ±â€¯0.04 °C (Setup 1) to 0.38 °C ±â€¯0.06 °C (Setup 3) (p=0.6) and from 87.0%±1.6% (Setup 3) to 88.1%±1.6% (Setup 2) (p=0.58), respectively. Both setups (1 and 2) with the MW generator outside the MR scanner room were beneficial to reduce electromagnetic interference during MWA. Moreover, provided that a shielded cable is utilized in setups 2 and 3, all configurations displayed negligible differences in temperature precision and DSCs, indicating that the location of the MW generator does not significantly impact the accuracy of thermometry during MWA.

Immune correlates of protection of the four-segmented Rift Valley fever virus candidate vaccine in mice.

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease caused by RVF virus (RVFV). RVFV infections in humans are usually asymptomatic or associated with mild febrile illness, although more severe cases of haemorrhagic disease and encephalitis with high mortality also occur. Currently, there are no licensed human vaccines available. The safety and efficacy of a genetically engineered four-segmented RVFV variant (hRVFV-4s) as a potential live-attenuated human vaccine has been tested successfully in mice, ruminants, and marmosets though the correlates of protection of this vaccine are still largely unknown. In the present study, we have assessed hRVFV-4s-induced humoral and cellular immunity in a mouse model of RVFV infection. Our results confirm that a single dose of hRVFV-4s is highly efficient in protecting naïve mice from developing severe disease following intraperitoneal challenge with a highly virulent RVFV strain and data show that virus neutralizing (VN) serum antibody titres in a prime-boost regimen are significantly higher compared to the single dose. Subsequently, VN antibodies from prime-boost-vaccinated recipients were shown to be protective when transferred to naïve mice. In addition, hRVFV-4s vaccination induced a significant virus-specific T cell response as shown by IFN-γ ELISpot assay, though these T cells did not provide significant protection upon passive transfer to naïve recipient mice. Collectively, this study highlights hRVFV-4s-induced VN antibodies as a major correlate of protection against lethal RVFV infection.

Immunity to Tick-Borne Encephalitis Virus NS3 Protein Induced with a Recombinant Modified Vaccinia Virus Ankara Fails to Afford Mice Protection against TBEV Infection.

Tick-borne encephalitis (TBE) is a serious neurological disease caused by TBE virus (TBEV). Because antiviral treatment options are not available, vaccination is the key prophylactic measure against TBEV infections. Despite the availability of effective vaccines, cases of vaccination breakthrough infections have been reported. The multienzymatic non-structural protein 3 (NS3) of orthoflaviviruses plays an important role in polyprotein processing and virus replication. In the present study, we evaluated NS3 of TBEV as a potential vaccine target for the induction of protective immunity. To this end, a recombinant modified vaccinia virus Ankara that drives the expression of the TBEV NS3 gene (MVA-NS3) was constructed. MVA-NS3 was used to immunize C57BL/6 mice. It induced NS3-specific immune responses, in particular T cell responses, especially against the helicase domain of NS3. However, MVA-NS3-immunized mice were not protected from subsequent challenge infection with a lethal dose of the TBEV strain Neudoerfl, indicating that in contrast to immunity to prME and NS1, NS3-specific immunity is not an independent correlate of protection against TBEV in this mouse model.

Predicting absolute aqueous solubility by applying a machine learning model for an artificially liquid-state as proxy for the solid-state.

In this study, we use machine learning algorithms with QM-derived COSMO-RS descriptors, along with Morgan fingerprints, to predict the absolute solubility of drug-like compounds. The QM-derived descriptors account for the molecular properties of the solute, i.e., the solute-solute interactions in an artificial-liquid-state (super-cooled liquid), and the solute-solvent interactions in solution. We employ two main approaches to predict solubility: (i) a hypothetical pathway that involves melting the solute at room temperature T = T¯ ([Formula: see text]) and mixing the artificially liquid solute into the solvent ([Formula: see text]). In this approach [Formula: see text] is predicted using machine learning models, and the [Formula: see text] is obtained from COSMO-RS calculations; (ii) direct solubility prediction using machine learning algorithms. The models were trained on a large number of Bayer in-house compounds for which water solubility data is available at physiological pH of 6.5 and ambient temperature. We also evaluated our models using external datasets from a solubility challenge. Our models present great improvements compared to the absolute solubility prediction with the QSAR model for the artificial liquid state as implemented in the COSMOtherm software, for both in-house and external datasets. We are furthermore able to demonstrate the superiority of QM-derived descriptors compared to cheminformatics descriptors. We finally present low-cost alternative models using fragment-based COSMOquick calculations with only marginal reduction in the quality of predicted solubility.

Induction of humoral and cell-mediated immunity to the NS1 protein of TBEV with recombinant Influenza virus and MVA affords partial protection against lethal TBEV infection in mice.

Introduction: Tick-borne encephalitis virus (TBEV) is one of the most relevant tick-transmitted neurotropic arboviruses in Europe and Asia and the causative agent of tick-borne encephalitis (TBE). Annually more than 10,000 TBE cases are reported despite having vaccines available. In Europe, the vaccines FSME-IMMUN® and Encepur® based on formaldehyde-inactivated whole viruses are licensed. However, demanding vaccination schedules contribute to sub-optimal vaccination uptake and breakthrough infections have been reported repeatedly. Due to its immunogenic properties as well as its role in viral replication and disease pathogenesis, the non-structural protein 1 (NS1) of flaviviruses has become of interest for non-virion based flavivirus vaccine candidates in recent years. Methods: Therefore, immunogenicity and protective efficacy of TBEV NS1 expressed by neuraminidase (NA)-deficient Influenza A virus (IAV) or Modified Vaccinia virus Ankara (MVA) vectors were investigated in this study. Results: With these recombinant viral vectors TBEV NS1-specific antibody and T cell responses were induced. Upon heterologous prime/boost regimens partial protection against lethal TBEV challenge infection was afforded in mice. Discussion: This supports the inclusion of NS1 as a vaccine component in next generation TBEV vaccines.

A recombinant Modified Vaccinia virus Ankara expressing prME of tick-borne encephalitis virus affords mice full protection against TBEV infection.

Introduction: Tick-borne encephalitis virus (TBEV) is an important human pathogen that can cause a serious disease involving the central nervous system (tick-borne encephalitis, TBE). Although approved inactivated vaccines are available, the number of TBE cases is rising, and breakthrough infections in fully vaccinated subjects have been reported in recent years. Methods: In the present study, we generated and characterized a recombinant Modified Vaccinia virus Ankara (MVA) for the delivery of the pre-membrane (prM) and envelope (E) proteins of TBEV (MVA-prME). Results: MVA-prME was tested in mice in comparison with a licensed vaccine FSME-IMMUN® and proved to be highly immunogenic and afforded full protection against challenge infection with TBEV. Discussion: Our data indicate that MVA-prME holds promise as an improved next-generation vaccine for the prevention of TBE.

A Real-Time Energy Monitoring System for an MRI Hybrid Ablation System.

The MRI hybrid ablation system is an approach to use the MR (magnetic resonance) scanner's radiofrequency amplifier itself as power source for ablation. Hereby, an electrode is connected to the MR internal radiofrequency amplifier. An average RF power is provided through a train of short RF pulses, which is sufficient to thermally destroy tissue. However, ablation with too high power values can cause tissue carbonizations, thus impeding the ablation procedure. Therefore, monitoring of the energy and the power absorbed inside the tissue is necessary. For this purpose, a measurement system was designed to measure the energy applied to the tissue when using the concept of an MRI hybrid ablation system. The system consists of a dual-directional coupler, RF-to-RMS sensors, and a microcontroller. The gradient calculation of the measured energy curve provides information about the absorbed RF power in the tissue. Validation measurements of the system were performed and compared with measurements from the MR-internal power measurement system. The energy monitoring system was also tested in an ablation experiment with ex-vivo animal tissue using the MRI hybrid ablation system. The measurements showed that the applied RF power can be monitored in real-time. It has been shown that the mean RF power absorbed in the patient decreased during an ablation procedure due to an occurring impedance mismatch and tissue changes. In further work, the influence of the monitoring system on the quality of the MR images should be investigated. Clinical relevance- This paper demonstrates an energy monitoring system for an RF ablation system, which can be used inside an MR environment.

Comparison study of reconstruction algorithms for volumetric necrosis maps from 2D multi-slice GRE thermometry images.

Cancer is a disease which requires a significant amount of careful medical attention. For minimally-invasive thermal ablation procedures, the monitoring of heat distribution is one of the biggest challenges. In this work, three approaches for volumetric heat map reconstruction (Delauney triangulation, minimum volume enclosing ellipsoids (MVEE) and splines) are presented based on uniformly distributed 2D MRI phase images rotated around the applicator's main axis. We compare them with our previous temperature interpolation method with respect to accuracy, robustness and adaptability. All approaches are evaluated during MWA treatment on the same data sets consisting of 13 ex vivo bio protein phantoms, including six phantoms with simulated heat sink effects. Regarding accuracy, the DSC similarity results show a strong trend towards the MVEE ([Formula: see text]) and the splines ([Formula: see text]) method compared to the Delauney triangulation ([Formula: see text]) or the temperature interpolation ([Formula: see text]). Robustness is increased for all three approaches and the adaptability shows a significant trend towards the initial interpolation method and the splines. To overcome local inhomogeneities in the acquired data, the use of adaptive simulations should be considered in the future. In addition, the transfer to in vivo animal experiments should be considered to test for clinical applicability.

Infections with highly pathogenic avian influenza A virus (HPAIV) H5N8 in harbor seals at the German North Sea coast, 2021.

In brain tissue of three harbor seals of the German North Sea coast, high virus loads of highly pathogenic avian influenza virus (HPAIV) H5N8 were detected. Identification of different virus variants indicates high exposure to HPAIV circulating in wild birds, but there is no evidence for H5 specific antibodies in healthy seals. Replication of avian viruses in seals may allow HPAIV to acquire mutations needed to adapt to mammalian hosts as shown by PB2 627K variants detected in these cases.

3D-Printed Floating Cable Traps for MRI guided Microwave Ablation.

Magnetic Resonance Imaging (MRI) guided Microwave Ablation (MWA) allows for real-time therapy monitoring with MRI-thermometry. The MWA generator emits Radio Frequency (RF) interference, which can limit the accuracy of therapy monitoring. The image quality is enhanced by Floating Cable Traps (FCTs) that are used to attenuate common mode currents on supply lines between a MWA generator, and its ablation applicator. The effect of an FCT on the Signal to Noise Ratio (SNR), and changes in the MRI spectrum are discussed in this paper. The application of FCT can bring significant improvements in both, the MRI spectrum and the SNR.Floating Cable Traps are user-friendly. FCT enable coaxial cables to reduce interferences emitted in MRI guided interventions. It is used to selectively attenuate frequencies in the MRI's range. This can increase the image's Signal to Noise Ratio.

Design of a System for Magnetic-Resonance-Guided Irreversible Electroporation.

Irreversible electroporation (IRE) is a non-thermal tumor ablation method where strong electrical fields between at least two electrodes are used and can be seen as an alternative to thermal ablation techniques. The therapy outcome directly dependents on the position of the electrodes. Real-time monitoring of the IRE by magnetic resonance imaging (MRI) would allow to detect unwanted electrode displacement and to apply visualization methods for the ablation area. This requires that the IRE generator does not significantly interfere with the MRI. Currently, there is no IRE generator available designed for MRI-guided IRE.This paper presents an IRE system specifically developed for use in an MRI environment. The system is initially tested with a standard IRE sequence and then the interference between a clinical 3 T MRI device and the IRE system is investigated using a noise measurement and the signal-to-noise ratio (SNR) of images acquired with a gradient echo (GRE) sequence. The results show, that although the SNR of the images decrease by maximal 36 % when the IRE system is switched on, image quality does not visibly degrade. Hence, MRI-guided IRE is feasible with the proposed system.Clinical relevance- This paper demonstrates the possibility of MRI-guided IRE with only minor image degradation when the IRE system is used in parallel with MRI imaging.

Older adults lack SARS CoV-2 cross-reactive T lymphocytes directed to human coronaviruses OC43 and NL63.

Currently, infections with SARS-Coronavirus-2 (SARS-CoV-2), the causative agent of the COVID-19 pandemic, are responsible for substantial morbidity and mortality worldwide. Older adults subjects > 60 years of age account for > 95% of the over one million fatal cases reported to date. It is unclear why in this age group SARS-CoV-2 infection causes more severe disease than in young adults. We hypothesized that differences in SARS-CoV-2 cross-reactive cellular immunity induced after infection with human coronaviruses (HCoVs), like OC43 and NL63, were at the basis of the differential mortality (and morbidity) observed after SARS-CoV-2 infection, because a small proportion of HCoV-specific T cells cross-react with SARS-CoV-2. Our data demonstrate that pre-existing T cell immunity induced by circulating human alpha- and beta-HCoVs is present in young adult individuals, but virtually absent in older adult subjects. Consequently, the frequency of cross-reactive T cells directed to the novel pandemic SARS-CoV-2 was minimal in most older adults. To the best of our knowledge, this is the first time that the presence of cross-reactive T cells to SARS-CoV-2 is compared in young and older adults. Our findings provide at least a partial explanation for the more severe clinical outcome of SARS-CoV-2 infection observed in the elderly. Moreover, this information could help to design efficacious vaccines for this age group, aiming at the induction of cell-mediated immunity.

Tick-Borne Encephalitis Virus: A Quest for Better Vaccines against a Virus on the Rise.

Tick-borne encephalitis virus (TBEV), a member of the family Flaviviridae, is one of the most important tick-transmitted viruses in Europe and Asia. Being a neurotropic virus, TBEV causes infection of the central nervous system, leading to various (permanent) neurological disorders summarized as tick-borne encephalitis (TBE). The incidence of TBE cases has increased due to the expansion of TBEV and its vectors. Since antiviral treatment is lacking, vaccination against TBEV is the most important protective measure. However, vaccination coverage is relatively low and immunogenicity of the currently available vaccines is limited, which may account for the vaccine failures that are observed. Understanding the TBEV-specific correlates of protection is of pivotal importance for developing novel and improved TBEV vaccines. For affording robust protection against infection and development of TBE, vaccines should induce both humoral and cellular immunity. In this review, the adaptive immunity induced upon TBEV infection and vaccination as well as novel approaches to produce improved TBEV vaccines are discussed.

Influenza virus-specific CD4+ and CD8+ T cell-mediated immunity induced by infection and vaccination.

Influenza A and B virus infections are a major cause of respiratory disease in humans and are responsible for substantial morbidity and mortality worldwide. Vaccination against influenza mainly aims at the induction of virus neutralizing serum antibodies, which are an important correlate of protection provided that the antibodies match the strains causing the outbreaks antigenically. In addition, virus-specific T cells are known to contribute to protective immunity to influenza virus infections by limiting duration and severity of the disease. As the majority of virus-specific T cells recognize epitopes located in relatively conserved proteins, like the Nucleoprotein and Matrix 1 protein, they display a high degree of cross-reactivity with a wide range of influenza viruses, including newly emerging viruses of alternative subtypes. Advancing our understanding of influenza virus-specific T cell responses and their role in protective immunity against influenza will aid the rational design of novel vaccines that could induce robust, broad and long-lasting immune responses. Here, we discuss the contribution of influenza virus-specific CD4+ and CD8+ T cells to protective immunity against influenza infection and the requirements and strategies for their induction by natural infection or vaccination, especially in children.

Response Modifiers: Tweaking the Immune Response Against Influenza A Virus.

Despite causing pandemics and yearly epidemics that result in significant morbidity and mortality, our arsenal of options to treat influenza A virus (IAV) infections remains limited and is challenged by the virus itself. While vaccination is the preferred intervention strategy against influenza, its efficacy is reduced in the elderly and infants who are most susceptible to severe and/or fatal infections. In addition, antigenic variation of IAV complicates the production of efficacious vaccines. Similarly, effectiveness of currently used antiviral drugs is jeopardized by the development of resistance to these drugs. Like many viruses, IAV is reliant on host factors and signaling-pathways for its replication, which could potentially offer alternative options to treat infections. While host-factors have long been recognized as attractive therapeutic candidates against other viruses, only recently they have been targeted for development as IAV antivirals. Future strategies to combat IAV infections will most likely include approaches that alter host-virus interactions on the one hand or dampen harmful host immune responses on the other, with the use of biological response modifiers (BRMs). In principle, BRMs are biologically active agents including antibodies, small peptides, and/or other (small) molecules that can influence the immune response. BRMs are already being used in the clinic to treat malignancies and autoimmune diseases. Repurposing such agents would allow for accelerated use against severe and potentially fatal IAV infections. In this review, we will address the potential therapeutic use of different BRM classes to modulate the immune response induced after IAV infections.

Recombinant influenza A viruses as vaccine vectors.

INTRODUCTION: Various viruses, including poxviruses, adenoviruses and vesicular stomatitis virus, have been considered as vaccine vectors for the delivery of antigens of interest in the development of vaccines against newly emerging pathogens. AREAS COVERED: Here, we review results that have been obtained with influenza A viruses (IAV) as vaccine vectors. With the advent of reverse genetics technology, IAV-based recombinant vaccine candidates have been constructed that induce protective immunity to a variety of different pathogens of interest, including West Nile virus, Plasmodium falciparum and respiratory syncytial virus. The various cloning strategies to produce effective and attenuated, safe to use IAV-based viral vectors are discussed. EXPERT COMMENTARY: It was concluded that IAV-based vector system has several advantages and holds promise for further development.

Setup of an Ablation Magnetic Resonance Imaging Hybrid System: Using MR Imaging Sequences to Destroy Tissue.

The need of external ablation generators complicates the setup of magnetic resonance (MR) guided interventions, e. g. due to inserting devices with ferrite components into the MR room or because of image distortions due to RF interferences. By using the power provided from the MR internal power amplifier, it is possible to avoid external ablation generators. Using imaging sequences with a high duty cycle, a sufficient mean power value can be generated to destroy tissue. In this paper, it has been shown that it is possible to destroy tissue with such an ablation MRI hybrid system. Simulations were done to calculate the specific absorption rate (SAR) generated by an ablation electrode at the Larmor frequency for a 3 T MR device. The SAR values were then compared with ablation experiments performed inside an MR device with protein phantoms.

Broadly protective influenza vaccines: design and production platforms.

Effective vaccines are the cornerstone of our defenses against acute influenza virus infections that result in ∼500 000 annual deaths worldwide. For decades, an on-going concerted effort has been to develop a universal influenza vaccine to combat the looming threat of potentially pandemic emerging and re-emerging influenza viruses. To address the need for rapid efficacious vaccines that could mitigate the impact of seasonal and future pandemics, multiple platforms are under development and/or investigation. What is clear is that any universal vaccine must provide long-lasting cross-protective immunity that can induce both B and T cell responses. This review will explore some of the universal influenza vaccine platforms in the contexts of their ability to induce long-lasting and cross-protective T cell immunity.

Influenza vaccines: 'tailor-made' or 'one fits all'.

Currently used inactivated influenza vaccines aim at the induction of virus-neutralizing antibodies directed to the variable head domain of the viral hemagglutinin. Although these vaccines are effective against antigenically matching virus strains, they offer little protection against antigenically distinct drift variants or potentially pandemic viruses of alternative subtypes. In the last decades, the threat of novel influenza pandemics has sparked research efforts to develop vaccines that induce more broadly protective immunity. Here, we discuss the immune responses induced by conventional 'tailor-made' inactivated and live influenza vaccines and novel 'one fits all' candidate vaccines able to induce cross-reactive virus-specific antibody and T cell responses and to afford protection to a wider range of influenza viruses.