Cytotoxic immune cells do not affect TDP-43 and p62 sarcoplasmic aggregation but influence TDP-43 localisation

Sporadic inclusion body myositis (sIBM) is an idiopathic inflammatory myopathy with invasion of CD8 T cells in muscle and aggregation of proteins in the sarcoplasm. TDP-43 and p62 are two proteins that aggregate in affected muscle, and have been suggested as specific markers for sIBM over other inflammatory myopathies. TDP-43 is also mislocalised from the nucleus to the sarcoplasm in sIBM. It is not clear if inflammation precedes protein aggregation in sIBM. This study investigated if exposure to cytotoxic inflammatory cells caused TDP-43 and p62 aggregation or TDP-43 mislocalisation in cultured myotubes. TALL-104 coculture was highly cytotoxic to myotubes after 24 h. Secretion of IFNγ and TNFα were higher in cocultures compared to monocultured TALL-104 cells, indicating activation. TALL-104 cells attached to and infiltrated myotubes. There was no effect of TALL-104 coculture on TDP-43 or p62 sarcoplasmic aggregate size or frequency. However, there was decreased localisation of TDP-43 to the nucleus with TALL-104 coculture compared to control. In an in vitro setting, cytotoxic immune cells did not cause TDP-43 or p62 sarcoplasmic aggregation, suggesting cellular cytotoxicity may not trigger aggregation of these proteins. However TALL-104 coculture influenced TDP-43 localisation, suggesting cytotoxic immune cells may contribute to TDP-43 localisation shifts which is observed in sIBM

Influence of Inflammatory Cytokines IL-1β and IFNγ on Sarcoplasmic Aggregation of p62 and TDP-43 in Myotubes

Skeletal muscle of patients with sporadic inclusion body myositis (sIBM) presents with inflammation, including upregulation of inflammatory cytokines such as interferon γ (IFNγ). Non-inflammatory features are also observed, like the sarcoplasmic accumulation of proteins including TDP-43 and p62. This study aimed to investigate the effect of IFNγ and interleukin 1-β (IL-1β) on TDP-43 and p62 aggregation in vitro. Primary human myotubes were treated with IL-1β (20 ng/mL) and IFNγ (750 ng/mL) separately or combined for 48 hr. Sarcoplasmic TDP-43 aggregates and p62 puncta were assessed using image analysis for size, frequency, and colocalization with each other. Total protein expression of TDP-43, p62 and LC3 was assessed using western blotting. The subcellular localization of TDP-43 was also analyzed using image analysis. Combined IL-1β and IFNγ treatment increased puncta size of p62 compared to control (0.49 ± 0.13 µm2 versus 0.28 ± 0.06 µm2), without affecting puncta frequency or p62 expression but with an increased LC3II/LC3I ratio, suggesting autophagic alterations. IL-1β or IFNγ did not alter p62 puncta size or frequency, suggesting a combined insult of multiple inflammatory mediators is necessary to cause p62 alterations. IL-1β increased p62 protein expression in an autophagy-independent manner. None of the cytokine treatments affected TDP-43 protein expression, size, or frequency of TDP-43 aggregates or localization, suggesting IL-1β and IFNγ may influence TDP-43 processing in human skeletal muscle cells. TDP-43 was localized to the sarcoplasm under control conditions, suggesting this may not be a pathological feature. Overall, sIBM-like TDP-43/p62 features were not triggered by IL-1β and/or IFNγ

Production and Utility of Extracellular Vesicles with 3D Culture Methods

In recent years, extracellular vesicles (EVs) have emerged as promising biomarkers, cell-free therapeutic agents, and drug delivery carriers. Despite their great clinical potential, poor yield and unscalable production of EVs remain significant challenges. When using 3D culture methods, such as scaffolds and bioreactors, large numbers of cells can be expanded and the cell environment can be manipulated to control the cell phenotype. This has been employed to successfully increase the production of EVs as well as to enhance their therapeutic effects. The physiological relevance of 3D cultures, such as spheroids, has also provided a strategy for understanding the role of EVs in the pathogenesis of several diseases and to evaluate their role as tools to deliver drugs. Additionally, 3D culture methods can encapsulate EVs to achieve more sustained therapeutic effects as well as prevent premature clearance of EVs to enable more localised delivery and concentrated exosome dosage. This review highlights the opportunities and drawbacks of different 3D culture methods and their use in EV research

Monitoring Biosensor Activity in Living Cells with Fluorescence Lifetime Imaging Microscopy

Live-cell microscopy is now routinely used to monitor the activities of the genetically encoded biosensor proteins that are designed to directly measure specific cell signaling events inside cells, tissues, or organisms. Most fluorescent biosensor proteins rely on Förster resonance energy transfer (FRET) to report conformational changes in the protein that occur in response to signaling events, and this is commonly measured with intensity-based ratiometric imaging methods. An alternative method for monitoring the activities of the FRET-based biosensor proteins is fluorescence lifetime imaging microscopy (FLIM). FLIM measurements are made in the time domain, and are not affected by factors that commonly limit intensity measurements. In this review, we describe the use of the digital frequency domain (FD) FLIM method for the analysis of FRET signals. We illustrate the methods necessary for the calibration of the FD FLIM system, and demonstrate the analysis of data obtained from cells expressing “FRET standard” fusion proteins. We then use the FLIM-FRET approach to monitor the changes in activities of two different biosensor proteins in specific regions of single living cells. Importantly, the factors required for the accurate determination and reproducibility of lifetime measurements are described in detail

Cell shape characteristics of human skeletal muscle cells as a predictor of myogenic competency: A new paradigm towards precision cell therapy

Skeletal muscle-derived cells (SMDC) hold tremendous potential for replenishing dysfunctional muscle lost due to disease or trauma. Current therapeutic usage of SMDC relies on harvesting autologous cells from muscle biopsies that are subsequently expanded in vitro before re-implantation into the patient. Heterogeneity can arise from multiple factors including quality of the starting biopsy, age and comorbidity affecting the processed SMDC. Quality attributes intended for clinical use often focus on minimum levels of myogenic cell marker expression. Such approaches do not evaluate the likelihood of SMDC to differentiate and form myofibres when implanted in vivo, which ultimately determines the likelihood of muscle regeneration. Predicting the therapeutic potency of SMDC in vitro prior to implantation is key to developing successful therapeutics in regenerative medicine and reducing implementation costs. Here, we report on the development of a novel SMDC profiling tool to examine populations of cells in vitro derived from different donors. We developed an image-based pipeline to quantify morphological features and extracted cell shape descriptors. We investigated whether these could predict heterogeneity in the formation of myotubes and correlate with the myogenic fusion index. Several of the early cell shape characteristics were found to negatively correlate with the fusion index. These included total area occupied by cells, area shape, bounding box area, compactness, equivalent diameter, minimum ferret diameter, minor axis length and perimeter of SMDC at 24 h after initiating culture. The information extracted with our approach indicates live cell imaging can detect a range of cell phenotypes based on cell-shape alone and preserving cell integrity could be used to predict propensity to form myotubes in vitro and functional tissue in vivo

Recent developments in targeting access to high cost medicines in Australia

BACKGROUND: In Australia, the Pharmaceutical Benefits Scheme (PBS) has developed a set of arrangements to control access to high-cost medicines to ensure their use is cost-effective. These medicines include the tumour necrosis factor-alpha inhibitors (TNFIs) for the treatment of rheumatoid arthritis. The aim of this first phase of a qualitative study was to explore basic views on the restricted access to TNFIs in order to confirm where further investigation should take place in the next phase. METHODS: Semi-structured interviews were conducted in 2004 with a member of the four relevant stakeholder groups. Participants were asked their opinions about features of the establishment, process and effects of the system of restricted access to TNFIs. Views on the collaboration between stakeholder groups in the decision-making process were also collected. RESULTS: The principle of 'controlled access' to TNFIs was supported in general. There were concerns regarding some of the specific eligibility criteria. Wider and more transparent stakeholder consultation was judged desirable. Some flexibility around prescribing of TNFIs by physicians, and regular review of the arrangements were proposed. These themes will inform the next phase of the study. CONCLUSION: This first phase highlighted a range of issues associated with the PBS arrangements restricting access to TNFIs. Timely review and report of issues and concerns associated with such policy developments that arose in practice are essential. There is a need for a more comprehensive exploration across a wide range of stakeholders with different perspectives that will in turn be helpful in guiding policy and practice around national arrangements to manage access to high-cost medicines

Role of L2 cysteines in papillomavirus infection and neutralization

Vaccination of mice with minor capsid protein L2 or passive transfer with the L2-specific neutralizing monoclonal antibody RG-1 protects against human papillomavirus type 16 (HPV16) challenge. Here we explored the nature of the RG-1 epitope and its contribution to viral infectivity. RG-1 bound equivalently HPV16 L2 residues 17-36 with or without an intact C22-C28 disulphide bridge. HPV16 L2 mutations K20A, C22A, C22S, C28A, C28S, or P29A prevented RG-1 binding, whereas Y19A, K23A or Q24A had no impact. Mutation of either C22 or C28 to alanine or serine compromises HPV16 pseudoviral infectivity both in vitro and in the murine vaginal tract, but does not impact pseudovirion assembly. Despite their lack of infectivity, HPV16 pseudovirions containing C22S or C28S mutant L2 bind to cell surfaces, are taken up, and expose the 17-36 region on the virion surface as for wild type HPV16 pseudovirions suggesting normal furin cleavage of L2. Mutation of the second cysteine residue in Bovine papillomavirus type 1 (BPV1) L2 to serine (C25S) dramatically reduced the infectivity of BPV1 pseudovirions. Surprisingly, in contrast to the double mutation in HPV16 L2, the BPV1 L2 C19S, C25S double mutation reduced BPV1 pseudovirion infectivity of 293TT cells by only half

Bioactive Materials

Bioactive substantially silica-free glass material with a hydroxyapatite layer thereon is described, as well as methods for producing hydroxyapatite on a borate glass

A simple and robust method for pre-wetting poly (lactic-co-glycolic) acid microspheres

Poly (lactic-co-glycolic) acid microspheres are amenable to a number of biomedical procedures that support delivery of cells, drugs, peptides or genes. Hydrophilisation or wetting of poly (lactic-co-glycolic) acid are an important pre-requisites for attachment of cells and can be achieved via exposure to plasma oxygen or nitrogen, surface hydrolysis with NaOH or chloric acid, immersion in ethanol and water, or prolonged incubation in phosphate buffered saline or cell culture medium. The aim of this study is to develop a simple method for wetting poly (lactic-co-glycolic) acid microspheres for cell delivery applications. A one-step ethanol immersion process that involved addition of serum-supplemented medium and ethanol to PLGA microspheres over 30 min–24 h is described in the present study. This protocol presents a more efficient methodology than conventional two-step wetting procedures. Attachment of human skeletal myoblasts to poly (lactic-co-glycolic) acid microspheres was dependent on extent of wetting, changes in surface topography mediated by ethanol pre-wetting and serum protein adsorption. Ethanol, at 70% (v/v) and 100%, facilitated similar levels of wetting. Wetting with 35% (v/v) ethanol was only achieved after 24 h. Pre-wetting (over 3 h) with 70% (v/v) ethanol allowed significantly greater (p ≤ 0.01) serum protein adsorption to microspheres than wetting with 35% (v/v) ethanol. On serum protein-loaded microspheres, greater numbers of myoblasts attached to constructs wetted with 70% ethanol than those partially wetted with 35% (v/v) ethanol. Microspheres treated with 70% (v/v) ethanol presented a more rugose surface than those treated with 35% (v/v) ethanol, indicating that more efficient myoblast adhesion to the former may be at least partially attributed to differences in surface structure. We conclude that our novel protocol for pre-wetting poly (lactic-co-glycolic) acid microspheres that incorporates biochemical and structural features into this biomaterial can facilitate myoblast delivery for use in clinical settings.This project was supported by grants from the UK Medical Research Council (MR/L002752/1) and Sir Halley Stewart Trust. The research was undertaken at UCL/UCLH which receives funding from the Department of Health’s NIHR as a Comprehensive Biomedical Research Centre.Published versio

Fourth-Generation Fan Assessment Numeration System (FANS) Design and Performance Specifications

The Fan Assessment Numeration System (FANS) is a measurement device for generating ventilation fan performance curves. Three different-sized FANS currently exist for assessing ventilation fans commonly used in poultry and livestock housing systems. All FANS consist of an array of anemometers inside an aluminum shroud that traverse the inlet or outlet of a ventilation fan. The FANS design has been updated several times since its inception and is currently in its fourth-generation (G4). The current design iteration (FANS-G4) is reported in this article with an emphasis on the hardware and software control, data acquisition systems, and operational reliability. Six FANS-G4 units were fabricated at the University of Kentucky (UK) Agricultural Machinery Research Laboratory and calibrated at the University of Illinois Urbana-Champaign (UIUC) Bioenvironmental and Structural Systems (BESS) Laboratory. Results demonstrated that the FANS-G4 was capable of measuring volumetric airflow to within 0.6% of full-scale (FS), which ranged from 15,000 to 56,000 m3 h-1