Clinical Features and Voxel-Based-Symptom-Lesion Mapping of Silent Aspiration in Acute Infratentorial Stroke.

Post-stroke dysphagia (PSD) is a severe and common complication after ischemic stroke. The role of silent aspiration as an important contributing factor in the development of a dysphagia-associated complications, in particular aspiration-associated pneumonia has been insufficiently understood. The aim of this study was to investigate the characteristics and risk factors of silent aspiration in patients with acute infratentorial stroke by FEES and to identify culprit lesions in stroke patient with a high risk of silent aspiration via voxel-based-symptom-lesion mapping (VBS/ML). This study is a retrospective observational study based on a prospectively collected FEES and stroke database. Consecutive patient cases with acute ischemic infratentorial stroke and FEES examination between 2017 and 2022 were identified. Group allocation was performed based on PAS scores. Imaging analysis was performed by manual assignment and by VBS/ML. Group comparisons were performed to assess silent aspiration characteristics. Binary logistic regression analysis was performed to determine if baseline clinical, demographic, and imaging parameters were helpful in predicting silent aspiration in patients. In this study 84 patient cases with acute infratentorial stroke who underwent FEES examination were included. Patients were moderately affected at admission (mean NIH-SS score at admission 5.7 SD ± 4.7). Most lesions were found pontine. Overall 40.5% of patients suffered from silent aspiration, most frequently in case of bilateral lesions. Patients with silent aspiration had higher NIH-SS scores at admission (p < 0.05), had a more severe swallowing disorder (p < 0.05) and were 4.7 times more likely to develop post-stroke pneumonia. Patients who underwent FEES examination later than 72 h after symptom onset were significantly more likely to suffer from silent aspiration and to develop pneumonia compared to patients who underwent FEES examination within the first 72 h (p < 0.05). A binary logistic regression model identified NIH-SS at admission as a weak predictor of silent aspiration. Neither in manual assignment of the lesions to brain regions nor in voxel-wise statistic regression any specific region was useful in prediction of silent aspiration. Silent aspiration is common in patients with infratentorial stroke and contributes to the risk for pneumonia. Patients with silent aspiration are more severely affected by stroke, but cannot reliably be identified by NIH-SS at admission or lesion location. Patients suffering from acute infratentorial stroke should been screened and examined for PSD and silent aspiration.

Optimization of lentiviral vector production for scale-up in fixed-bed bioreactor.

Lentiviral vectors (LVs) are promising tools for gene therapy. However, scaling up the production methods of LVs in order to produce high-quality vectors for clinical purposes has proven to be difficult. In this article, we present a scalable and efficient method to produce LVs with transient transfection of adherent 293T cells in a fixed-bed bioreactor. The disposable iCELLis bioreactors are scalable with a large three-dimensional (3D) growth area range between 0.53 and 500 m2, an integrated perfusion system, and a controllable environment for production. In this study, iCELLis Nano (2.67-4 m2) was used for optimizing production parameters for scale-up. Transfections were first done using traditional calcium phosphate method, but in later runs polyethylenimine was found to be more reliable and easier to use. For scalable LV production, perfusion rate control by measuring cell metabolite concentrations in the bioreactor leads to higher productivity and reduced costs. Optimization of cell seeding density for targeted cell concentration during transfection, use of low compaction fixed-bed and lowering the culture pH have a positive effect on LV productivity. These results show for the first time that iCELLis bioreactor is scalable from bench level to clinical scale LV production.

Production and purification of lentiviral vectors generated in 293T suspension cells with baculoviral vectors.

Lentivirus can be engineered to be a highly potent vector for gene therapy applications. However, generation of clinical grade vectors in enough quantities for therapeutic use is still troublesome and limits the preclinical and clinical experiments. As a first step to solve this unmet need we recently introduced a baculovirus-based production system for lentiviral vector (LV) production using adherent cells. Herein, we have adapted and optimized the production of these vectors to a suspension cell culture system using recombinant baculoviruses delivering all elements required for a safe latest generation LV preparation. High-titer LV stocks were achieved in 293T cells grown in suspension. Produced viruses were accurately characterized and the functionality was also tested in vivo. Produced viruses were compared with viruses produced by calcium phosphate transfection method in adherent cells and polyethylenimine transfection method in suspension cells. Furthermore, a scalable and cost-effective capture purification step was developed based on a diethylaminoethyl monolithic column capable of removing most of the baculoviruses from the LV pool with 65% recovery.

(Strept)avidin-displaying lentiviruses as versatile tools for targeting and dual imaging of gene delivery.

Lentiviruses have shown great promise for human gene therapy. However, no optimal strategies are yet available for noninvasive imaging of virus biodistribution and subsequent transduction in vivo. We have developed a dual-imaging strategy based on avidin-biotin system allowing easy exchange of the surface ligand on HIV-derived lentivirus envelope. This was achieved by displaying avidin or streptavidin fused to the transmembrane anchor of vesicular stomatitis virus G protein on gp64-pseudotyped envelopes. Avidin and streptavidin were efficiently incorporated on virus particles, which consequently showed binding to biotin in ELISA. These vectors, conjugated to biotinylated radionuclides and engineered to express a ferritin transgene, enabled for the first-time dual imaging of virus biodistribution and transduction pattern by single-photon emission computed tomography and magnetic resonance imaging after stereotactic injection into rat brain. In addition, vector retargeting to cancer cells overexpressing CD46, epidermal growth factor and transferrin receptors using biotinylated ligands and antibodies was demonstrated in vitro. In conclusion, we have generated novel lentivirus vectors for noninvasive imaging and targeting of lentivirus-mediated gene delivery. This study suggests that these novel vectors could be applicable for the treatment of central nervous system disorders and cancer.

Generation of lentivirus vectors using recombinant baculoviruses.

In spite of advances in conventional four-plasmid transient transfection methods and development of inducible stable production cell lines, production of replication-defective lentiviral vectors in clinical scale has been challenging. Baculovirus technology offers an alternative to scalable virus production as a result of fast and easy production of baculoviruses, efficient transduction of mammalian cells and safety of the baculoviruses. As a first step toward scalable lentiviral production system, we have constructed four recombinant baculoviruses: the BAC-transfer virus expresses green fluorescent protein (GFP) as a transgene and BAC-gag-pol, BAC-vesicular stomatitis virus glycoprotein G and BAC-rev express all elements required for a safe lentivirus vector generation. Following 293T cell transduction with recombinant baculoviruses functional lentiviruses were produced. Different baculovirus concentrations, mediums and transduction times were used to find optimal conditions for lentivirus production. The unconcentrated lentiviral titers in cell culture mediums were on average 2.5 x 10(6) TU ml(-1), which are comparable to titers of the lentiviruses produced by conventional four-plasmid methods. Lentiviruses produced by baculovirus method transduced HeLa cells and showed sustained GFP expression. No evidence of the formation of replication competent lentiviruses was detected by p24 enzyme-linked immunosorbent assay. Our results show that baculoviruses are an attractive alternative for the production of lentiviruses in mammalian cells.

Plain language for interpreting in consulting rooms.

Intercultural communication is by nature a complex activity. In a multilingual society like ours, it will inevitably surface in the health care sector. The services of an interpreter are often considered to break the impasse in this communication process. The communication problem between the two parties, the service provider and client/patient, is often not simply a matter of language but societal factors of which the liaison interpreter should be aware of also plays a major role for effective extended communication. This article focuses on some of the problems in rendering an oral source text in multilingual and multicultural societies such as South Africa in which there are heterogeneous target audiences for interpreting. It is pointed out that interpreters in such societies must take into account the heterogeneity of the target audiences, or otherwise interpreting will only be symbolic gestures, empty of value, and thus not communicate the message intended. In the process the limitations of the interpreter and how the presence of the interpreter can be facilitated, is also highlighted.

Density of Langerhans' cells in ATPase stained epidermal sheet preparations from stasis dermatitis skin of the lower leg.

The density of Langerhans' cells (LC) stained with ATPase was determined in epidermal sheet preparations in skin specimens from patients with stasis dermatitis of the lower leg. Their density was highest in areas adjacent to manifest stasis dermatitis areas. Clinically diseased skin contained less LC than the adjacent areas, but still more than skin from control patients without venous insufficiency. The increased number of ATPase positive cells within and around stasis dermatitis skin might contribute to the high number of contact allergies observed in this patient group because of the antigen presenting capacity of these cells.

Molecular probes: what is the range of their interaction with the environment?

We performed pressure-tuning hole-burning experiments on a modified cytochrome c protein in a glycerol/buffer glass. The shift and the broadening of the holes were investigated for various frequencies within the inhomogeneous band. On the basis of a simple model, we were able to estimate the interaction range between chromophore and protein. It is approximately 4.5 A. The parameters that enter the model are the compressibility, the static mean-square displacement, the inhomogeneous width, and the average spectral shift per pressure. From this result and from our experiments on pressure-induced denaturing, we conclude that water molecules have to be brought very close to the chromophore during the denaturation process.

The X-Ray Emission of the Centaurus A Jet.

The extended nonthermal X-ray emission of extragalactic jets like Centaurus A can only be explained by in situ particle acceleration. The only energy source in the entire jet region is the magnetic field. Magnetic reconnection can convert the free energy stored in the helical configuration to particle kinetic energy. In the collisionless magnetized jet plasma, the inertia-driven reconnection is operating in a highly filamentary magnetic flux rope, and this results in a continuously charged particle acceleration. The synchrotron radiation of these particles can cause the observed X-ray emission in Centaurus A.