Mast Cell Purification Protocols.

Studying a tissue-specific mast cell can be of particular benefit given the heterogeneity that is known to exist among mast cells isolated or developed from different sources. Methods for isolating mast cells from a variety of tissues have been in existence for a number of years although, over time, these methodologies have been refined. We have had considerable experience studying mast cells isolated from human lung tissue. It is for this reason that, in this chapter, we provide detailed methods for the isolation and purification of human lung mast cells. However, it should be noted that the methods that are described in this chapter are generally applicable to the isolation of mast cells from different tissues, and this will also be discussed.

HTA methodology and value frameworks for evaluation and policy making for cell and gene therapies.

This last decade has been marked by significant advances in the development of cell and gene (C&G) therapies, such as gene targeting or stem cell-based therapies. C&G therapies offer transformative benefits to patients but present a challenge to current health technology decision-making systems because they are typically reviewed when clinical efficacy data are very limited and when there is uncertainty about the long-term durability of outcomes. These challenges are not unique to C&G therapies, but they face more of these barriers, reflecting the need for adapting existing value assessment frameworks. Still, C&G therapies have the potential to be cost-effective even at very high price points. The impact on healthcare budgets will depend on the success rate of pipeline assets and on the extent to which C&G therapies will expand to wider pathologies beyond rare or ultra-rare diseases. Getting pricing and reimbursement models right is important for incentivising research and development investment while not jeopardising the sustainability of healthcare systems. Payers and manufacturers therefore need to acknowledge each other's constraints-limitations in the evidence generation on the manufacturer side, budget considerations on the payer side-and embrace innovative thinking and approaches to ensure timely delivery of therapies to patients. Several experts in health technology assessment and clinical experts have worked together to produce this publication and identify methodological and policy options to improve the assessment of C&G therapies, and make it happen better, faster and sustainably in the coming years.

Orai and TRPC channel characterization in FcεRI-mediated calcium signaling and mediator secretion in human mast cells.

Inappropriate activation of mast cells via the FcεRI receptor leads to the release of inflammatory mediators and symptoms of allergic disease. Calcium influx is a critical regulator of mast cell signaling and is required for exocytosis of preformed mediators and for synthesis of eicosanoids, cytokines and chemokines. Studies in rodent and human mast cells have identified Orai calcium channels as key contributors to FcεRI-initiated mediator release. However, until now the role of TRPC calcium channels in FcεRI-mediated human mast cell signaling has not been published. Here, we show evidence for the expression of Orai 1,2, and 3 and TRPC1 and 6 in primary human lung mast cells and the LAD2 human mast cell line but, we only find evidence of functional contribution of Orai and not TRPC channels to FcεRI-mediated calcium entry. Calcium imaging experiments, utilizing an Orai selective antagonist (Synta66) showed the contribution of Orai to FcεRI-mediated signaling in human mast cells. Although, the use of a TRPC3/6 selective antagonist and agonist (GSK-3503A and GSK-2934A, respectively) did not reveal evidence for TRPC6 contribution to FcεRI-mediated calcium signaling in human mast cells. Similarly, inactivation of STIM1-regulated TRPC1 in human mast cells (as tested by transfecting cells with STIM1-KK684-685EE - TRPC1 gating mutant) failed to alter FcεRI-mediated calcium signaling in LAD2 human mast cells. Mediator release assays confirm that FcεRI-mediated calcium influx through Orai is necessary for histamine and TNFα release but is differentially involved in the generation of cytokines and eicosanoids.

Mast cell purification protocols.

Studying a tissue-specific mast cell can be of particular benefit given the heterogeneity that is known to exist among mast cells isolated or developed from different sources. Methods for isolating mast cells from a variety of tissues have been in existence for a number of years although, over time, these methodologies have been refined. We have had considerable experience studying mast cells isolated from human lung tissue. It is for this reason that, in this chapter, we provide detailed methods for the isolation and purification of human lung mast cells. However, it should be noted that the methods that are described in this chapter are generally applicable to the isolation of mast cells from different tissues and this will also be discussed.