Dietary Nitrate Acutely and Markedly Increased Exhaled Nitric Oxide in a Cystic Fibrosis Case

Airway nitric oxide (NO) is a ubiquitous signaling molecule with bronchoprotective, antiinflammatory and anti-infective roles. Cystic fibrosis (CF) is a chronic lung condition associated with deceased exhaled NO. Strategies to increase exhaled NO in CF have yielded inconsistent results. A potential new method of increasing systemic NO involves ingestion of dietary, inorganic nitrate which is reduced to nitrite and NO. We present the case of a 12 year-old, athletic male with CF who demonstrated acute but marked increases in exhaled NO following dietary nitrate consumption compared to placebo

Proline pre-conditioning of Jurkat cells improves recovery after cryopreservation

Cell therapies such as allogenic CAR T-cell therapy, natural killer cell therapy and stem cell transplants must be cryopreserved for transport and storage. This is typically achieved by addition of dimethyl sulfoxide (DMSO) but the cryoprotectant does not result in 100% cell recovery. New additives or technologies to improve their cryopreservation could have major impact for these emerging therapies. L-Proline is an amino acid osmolyte produced as a cryoprotectant by several organisms such as the codling moth Cydia pomonella and the larvae of the fly Chymomyza costata, and has been found to modulate post-thaw outcomes for several cell lines but has not been studied with Jurkat cells, a T lymphocyte cell line. Here we investigate the effectiveness of L-proline compared to D-proline and L-alanine for the cryopreservation of Jurkat cells. It is shown that 24-hour pre-freezing incubation of Jurkat cells with 200 mM L-proline resulted in a modest increase in cell recovery post-thaw at high cell density, but a larger increase in recovery was observed at the lower cell densities. L-Alanine was as effective as L-proline at lower cell densities, and addition of L-proline to the cryopreservation media (without incubation) had no benefit. The pre-freeze incubation with L-proline led to significant reductions in cell proliferation supporting an intracellular, biochemical, mechanism of action which was shown to be cell-density dependent. Controls with D-proline were found to reduce post-thaw recovery attributed to osmotic stress as D-proline cannot enter the cells. Preliminary analysis of apoptosis/necrosis profiles by flow cytometry indicated that inhibition of apoptosis is not the primary mode of action. Overall, this supports the use of L-proline pre-conditioning to improve T-cell post-thaw recovery without needing any changes to cryopreservation solutions nor methods and hence is simple to implement

Fluticasone furoate/vilanterol (100/25; 200/25 μg) improves lung function in COPD: a randomised trial.

SummaryBackgroundOnce-daily combination treatment is an attractive maintenance therapy for COPD. However, the dose of inhaled corticosteroid to use in a once-daily combination is unknown. We compared two strengths of fluticasone furoate (FF) plus vilanterol (VI), the same strengths of the individual components, and placebo.MethodsMulticentre, randomised, 24-week, double-blind, placebo-controlled, parallel-group study in stable, moderate-to-severe COPD subjects (N = 1224). Subjects were randomised to FF/VI (200/25 μg; 100/25 μg), FF (200 μg; 100 μg), VI 25 μg, or placebo, once daily in the morning. Co-primary efficacy endpoints; 0–4 h weighted mean (wm) FEV1 on day 168, and change from baseline in trough (23–24 h post-dose) FEV1 on day 169. The primary safety objective was adverse events (AEs).ResultsThere was a statistically significant (p < 0.001) increase in wm FEV1 (209 ml) and trough FEV1 (131 ml) for FF/VI 200/25 μg vs. placebo; similar changes were seen for FF/VI 100/25 μg vs. placebo. Whereas the difference between FF/VI 200/25 μg and VI 25 μg in change from baseline trough FEV1 (32 ml) was not statistically significant (p = 0.224), the difference between FF/VI 200/25 μg and FF 200 μg for wm FEV1 (168 ml) was significantly different (p < 0.001). VI 25 μg significantly improved wm and trough FEV1 vs. placebo (209 ml and 131 ml, respectively). No increase was seen in on-treatment AEs or serious AEs (SAEs), with active therapy vs. placebo.ConclusionsFF/VI provides rapid and significant sustained improvement in FEV1 in subjects with moderate-to-severe COPD, which was not influenced by the dose of FF. These data suggest that FF/VI may offer clinical efficacy in COPD and warrants additional study.GSK study number: HZC112207.ClinicalTrials.gov: NCT01054885

Spatial considerations during cryopreservation of a large volume sample

AbstractThere have been relatively few studies on the implications of the physical conditions experienced by cells during large volume (litres) cryopreservation – most studies have focused on the problem of cryopreservation of smaller volumes, typically up to 2 ml.This study explores the effects of ice growth by progressive solidification, generally seen during larger scale cryopreservation, on encapsulated liver hepatocyte spheroids, and it develops a method to reliably sample different regions across the frozen cores of samples experiencing progressive solidification.These issues are examined in the context of a Bioartificial Liver Device which requires cryopreservation of a 2 L volume in a strict cylindrical geometry for optimal clinical delivery. Progressive solidification cannot be avoided in this arrangement. In such a system optimal cryoprotectant concentrations and cooling rates are known. However, applying these parameters to a large volume is challenging due to the thermal mass and subsequent thermal lag. The specific impact of this to the cryopreservation outcome is required.Under conditions of progressive solidification, the spatial location of Encapsulated Liver Spheroids had a strong impact on post-thaw recovery. Cells in areas first and last to solidify demonstrated significantly impaired post-thaw function, whereas areas solidifying through the majority of the process exhibited higher post-thaw outcome. It was also found that samples where the ice thawed more rapidly had greater post-thaw viability 24 h post-thaw (75.7 ± 3.9% and 62.0 ± 7.2% respectively).These findings have implications for the cryopreservation of large volumes with a rigid shape and for the cryopreservation of a Bioartificial Liver Device

Levelling the playing field: Exploring inequalities and exclusions with a community‐based football league for people with experience of mental distress

Introduction Sport workforce strategy in the United Kingdom (UK) has identified the occupational therapy profession as being ideally positioned to contribute to public health agendas relating to tackling physical inactivity amongst marginalised populations, such as disabled people and people with experience of mental distress. However, a robust understanding of the enablers, restrictions, and exclusions such groups encounter when seeking to participate in sport and physical activity is currently lacking. Methods This study aimed to gain an in-depth understanding of the different ways people with experience of mental distress talked about their participation in a community-based football league in England, in the UK. Nine people took part in this strand of a larger participatory action research (PAR) study, which used go-along interviews as the method of data collection. In alignment with PAR seeking to address power imbalances, the data from the go-along interviews were analysed through a Foucauldian lens using a collaboratively produced analytic framework. Findings Participants constructed the community-based football league as fostering feelings of purpose and belonging, against a backdrop of them describing experiencing stigma and exclusion when seeking to be active in their wider communities. They used the concept of occupational marginalisation to further interpret their situation. Conclusion Understanding why and how people participate in football extends beyond seeing it as an individual exercise to shared social lives and occupations. With this perspective, occupational therapists could address occupational marginalisation in partnership with community sports organisations, collaborating for wider social change beyond specialist services

Scaling up Cryopreservation from Cell Suspensions to Tissues: Challenges and Successes

This chapter covers the key physical, biological and practical challenges encountered when developing cryopreservation protocols for larger biological structures and examines areas where cryopreservation has been successful in scaling to larger structures. Results from techniques being used in attempts to overcome these challenges are reviewed together with the indicators for future development that arise from them. The scale-up of cryopreservation to tissues with diverse functions and cell types makes the control of freezing and thawing more challenging. Technology may—or may not—be available depending on the size of the material involved. To meet the challenge there must be innovation in technology, techniques and understanding of damage-limiting strategies. Diversity of cell structure, size, shape and expected function means a similarly diverse response to any imposed cryopreservation conditions and interaction with ice crystals. The increasing diffusion distances involved, and diversity of permeability properties, will affect solutes, solvents, heat and cryoprotectant (CPA) transfer and so add to the diversity of response. Constructing a single protocol for cryopreservation of a larger sample (organoids to whole organs) becomes a formidable challenge

Automated dry thawing of cryopreserved haematopoietic cells is not adversely influenced by cryostorage time, patient age or gender.

Cell therapies are becoming increasingly widely used, and their production and cryopreservation should take place under tightly controlled GMP conditions, with minimal batch-to-batch variation. One potential source of variation is in the thawing of cryopreserved samples, typically carried out in water baths. This study looks at an alternative, dry thawing, to minimise variability in the thawing of a cryopreserved cell therapy, and compares the cellular outcome on thaw. Factors such as storage time, patient age, and gender are considered in terms of cryopreservation and thawing outcomes. Cryopreserved leukapheresis samples from 41 donors, frozen by the same protocol and stored for up to 17 years, have been thawed using automated, water-free equipment and by conventional wet thawing using a water bath. Post-thaw viability, assessed by both trypan blue and flow cytometry, showed no significant differences between the techniques. Similarly, there was no negative effect of the duration of frozen storage, donor age at sample collection or donor gender on post-thaw viability using either thawing method. The implication of these results is that the cryopreservation protocol chosen initially remains robust and appropriate for use with a wide range of donors. The positive response of the samples to water-free thawing offers potential benefits for clinical situations by removing the subjective element inherent in water bath thawing and eliminating possible contamination issues