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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.

Inappropriate activation of mast cells via the Fcε RI 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 Fcε RI‐initiated mediator release. However, until now the role of TRPC calcium channels in Fcε RI‐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 Fcε RI‐mediated calcium entry. Calcium imaging experiments, utilizing an Orai selective antagonist (Synta66) showed the contribution of Orai to Fcε RI‐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 Fcε RI‐mediated calcium signaling in human mast cells. Similarly, inactivation of STIM1‐regulated TRPC1 in human mast cells (as tested by transfecting cells with STIM1‐KK 684‐685 EE ‐ TRPC1 gating mutant) failed to alter Fcε RI‐mediated calcium signaling in LAD2 human mast cells. Mediator release assays confirm that Fcε RI‐mediated calcium influx through Orai is necessary for histamine and TNF α release but is differentially involved in the generation of cytokines and eicosanoids.

Inappropriate activation of mast cells via the FceRI 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 FceRI-initiated mediator release. However, until now the role of TRPC calcium channels in FceRI-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 FceRI-mediated calcium entry. Calcium imaging experiments, utilizing an Orai selective antagonist (Synta66) showed the contribution of Orai to FceRI-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 FceRI-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 FceRI-mediated calcium signaling in LAD2 human mast cells. Mediator release assays confirm that FceRI-mediated calcium influx through Orai is necessary for histamine and TNFα release but is differentially involved in the generation of cytokines and eicosanoids.

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.

SummaryBackground Prostaglandin E2 (PGE2) has been shown to inhibit IgE-dependent histamine release from human lung mast cells. This effect of PGE2 is believed to be mediated by EP2 receptors. However, definitive evidence that this is the case has been lacking in the absence of EP2-selective antagonists. Moreover, recent evidence has suggested that PGE2 activates EP4 receptors to inhibit respiratory cell function. Objective The aim of this study was to determine the receptor by which PGE2 inhibits human lung mast cell responses by using recently developed potent and selective EP2 and EP4 receptor antagonists alongside other established EP receptor ligands. Methods The effects of non-selective (PGE2, misoprostol), EP2-selective (ONO-AE1-259, AH13205, butaprost-free acid) and EP4-selective (L-902,688, TCS251) agonists on IgE-dependent histamine release and cyclic-AMP generation in mast cells were determined. The effects of EP2-selective (PF-04418948, PF-04852946) and EP4-selective (CJ-042794, L-161,982) antagonists on PGE2 responses of mast cells were studied. The expression of EP receptor subtypes was determined by RT-PCR. Results Prostaglandin E2, EP2 agonists and EP4 agonists inhibited IgE-dependent histamine release from mast cells. PGE2 and EP2 agonists, but not EP4 agonists, increased cyclic-AMP levels in mast cells. EP4-selective antagonists did not affect the PGE2 inhibition of histamine release, whereas EP2-selective antagonists caused rightward shifts in the PGE2 concentration–response curves. RT-PCR studies indicated that mast cells expressed EP2 and EP4 receptors. Conclusions and Clinical Relevance Although human lung mast cells may express both EP2 and EP4 receptors, the principal mechanism by which PGE2 inhibits mediator release in mast cells is by activating EP2 receptors.

The best characterised calcium channel in immune cells is the store-operated calcium release activated calcium channel (CRAC); studies in heterologous expression systems and knockout mice have identified Orai and its regulator STIM1, an ER-resident calcium sensor, as the molecular constituents of CRAC. Members of the TRPC family are also likely to contribute to store-operated calcium entry (SOCE) in immune cells. The expression of Orai and TRPC channels in primary human mast cells has not yet been investigated, but is of key importance given the critical role these cells play in the pathophysiology of type-1 hypersensitivity reactions. Allergic activation occurs following cross-linking of the FceRI receptor by antigen-IgE complexes that, via PLC-γ signalling, leads to robust activation of SOCE. The resulting calcium signal stimulates secretion and production of pro-inflammatory mediators. The aim of this study was to characterize the biophysical and pharmacological properties of CRAC channels in human lung mast cells (HLMC) following SOCE.RT-PCR revealed mRNA expression for Orai1, Orai2, STIM1 and STIM2 but not Orai3 in HLMCs. Whole-cell patch clamp recordings of isolated, cultured HLMCs were carried out using a Cs-based internal solution, with added IP3 and BAPTA to activate SOCE. These conditions activated an inwardly rectifying current with Erev= 18±4mV (n=6), a value less positive than expected for a pure Orai/CRAC current. However, the current was potentiated in divalent-free solution 563.7±6% (n=6), as expected for Orai/CRAC currents, and was significantly attenuated 51.4±8% (n=6), by the Orai-specific inhibitor Synta66 (10μM) (Ng et al. 2008., Di Sabatino et al. 2009). Calcium imaging in HLMCs loaded with fura-2 AM showed that Synta66 also attenuated anti-IgE induced calcium signaling by 62.2%(n=14). Taken together the data suggest that both Orai and TRPC channels contribute to SOCE in HLMCs.

Mast cells are important targets for the treatment of allergic diseases and anaphylaxis. Inappropriate and chronic activation of mast cells via the IgE receptor leads to the release of a range of pre-stored and newly synthesized inflammatory mediators and the symptoms of disease. Calcium influx is a critical regulator of mast cell signaling, with influx through ion channels absolutely required not only for the exocytosis of preformed mediators but also to direct the synthesis of eicosanoids, cytokines and chemokines.Studies in rodent and human mast cells have identified STIM-regulated Orai channels to be key players in initiating calcium influx and degranulation in antigen-stimulated mast cells; in rodent mast cells, a role for TRPC channels is also emerging. Here we report evidence for a role of TRPC channels in human mast cell signaling. Using gene microarray analysis, we find evidence for expression of multiple TRP family members in primary human lung mast cells and LAD2 cells; the expression of TRPC1 and TRPC6 was further confirmed by immunocytochemistry. Single cell fura-2 imaging experiments, showed that barium could partially substitute for calcium to support influx following antigen- and thapsigargin stimulation, consistent with a contribution of non-selective TRPC-like channels to store operated calcium entry in human mast cells. To investigate the potential involvement of TRPC1 in STIM-regulated store operated calcium signaling in human mast cells, LAD2 cells were transfected with the STIM1 (684KK685) TRPC1 gating mutant. Expression of the mutant, however failed to alter calcium signaling in either thapsigargin or antigen-stimulated cells. Further pharmacological and molecular experiments are currently being performed to further evaluate the putative role of other TRPC channels in human mast cell signaling.