Regulation of temporal cytokine production by co-stimulation receptors in TCR-T cells is lost in CAR-T cells.

CD8+ T cells contribute to immune responses by producing cytokines when their T-cell receptors (TCRs) recognise peptide antigens on major-histocompability-complex class I. However, excessive cytokine production can be harmful. For example, cytokine release syndrome is a common toxicity observed in treatments that activate T cells, including chimeric antigen receptor (CAR)-T-cell therapy. While the engagement of costimulatory receptors is well known to enhance cytokine production, we have limited knowledge of their ability to regulate the kinetics of cytokine production by CAR-T cells. Here we compare early (0-12 h) and late (12-20 h) production of IFN-gg, IL-2, and TNF-a production by T cells stimulated via TCR or CARs in the presence or absence ligands for CD2, LFA-1, CD28, CD27, and 4-1BB. For T cells expressing TCRs and 1st-generation CARs, activation by antigen alone was sufficient to stimulate early cytokine production, while co-stimulation by CD2 and 4-1BB was required to maintain late cytokine production. In contrast, T cells expressing 2nd-generation CARs, which have intrinsic costimulatory signalling motifs, produce high levels of cytokines in both early and late periods in the absence of costimulatory receptor ligands. Losing the requirement for costimulation for sustained cytokine production may contribute to the effectiveness and/or toxicity of 2nd-generation CAR-T-cell therapy.

Using CombiCells, a platform for titration and combinatorial display of cell surface ligands, to study T-cell antigen sensitivity modulation by accessory receptors.

Understanding cellular decisions due to receptor-ligand interactions at cell-cell interfaces has been hampered by the difficulty of independently varying the surface density of multiple different ligands. Here, we express the synthetic binder protein SpyCatcher, designed to form spontaneous covalent bonds with interactors carrying a Spytag, on the cell surface. Using this, we show that addition of different concentrations and combinations of native Spytag-fused ligands allows for the combinatorial display of ligands on cells within minutes. We use this combinatorial display of cell surface ligands-called CombiCells-to assess T cell antigen sensitivity and the impact of T cell co-stimulation and co-inhibition receptors. We find that the T cell receptor (TCR) displayed greater sensitivity to peptides on major-histocompatibility complexes (pMHC) than synthetic chimeric antigen receptor (CARs) and bi-specific T cell engager (BiTEs) display to their target antigen, CD19. While TCR sensitivity was greatly enhanced by CD2/CD58 interactions, CAR sensitivity was primarily but more modestly enhanced by LFA-1/ICAM-1 interactions. Lastly, we show that PD-1/PD-L1 engagement inhibited T cell activation triggered solely by TCR/pMHC interactions, as well as the amplified activation induced by CD2 and CD28 co-stimulation. The ability to easily produce cells with different concentrations and combinations of ligands should accelerate the study of receptor-ligand interactions at cell-cell interfaces.

A cell culture system to model pharmacokinetics using adjustable-volume perfused mixing chambers.

The pharmacokinetic (PK) profile of a drug is an essential factor in determining its efficacy, yet it is often neglected during in vitro cell culture experiments. Here, we present a system in which standard well plate cultures may be "plugged in" and perfused with PK drug profiles. Timed drug boluses or infusions are passed through a mixing chamber that simulates the PK volume of distribution specific to the desired drug. The user-specified PK drug profile generated by the mixing chamber passes through the incubated well plate culture, exposing cells to in vivo-like PK drug dynamics. The effluent stream from the culture may then optionally be fractionated and collected by a fraction collector. This low-cost system requires no custom parts and perfuses up to six cultures in parallel. This paper demonstrates a range of PK profiles the system can produce using a tracer dye, describes how to find the correct mixing chamber volumes to mimic PK profiles of drugs of interest, and presents a study exploring the effects of differing PK exposure on a model of lymphoma treatment with chemotherapy.

The Tumor Invasion Paradox in Cancer Stem Cell-Driven Solid Tumors.

Cancer stem cells (CSCs) are key in understanding tumor growth and tumor progression. A counterintuitive effect of CSCs is the so-called tumor growth paradox: the effect where a tumor with a higher death rate may grow larger than a tumor with a lower death rate. Here we extend the modeling of the tumor growth paradox by including spatial structure and considering cancer invasion. Using agent-based modeling and a corresponding partial differential equation model, we demonstrate and prove mathematically a tumor invasion paradox: a larger cell death rate can lead to a faster invasion speed. We test this result on a generic hypothetical cancer with typical growth rates and typical treatment sensitivities. We find that the tumor invasion paradox may play a role for continuous and intermittent treatments, while it does not seem to be essential in fractionated treatments. It should be noted that no attempt was made to fit the model to a specific cancer, thus, our results are generic and theoretical.

Multi-Stream Perfusion Bioreactor Integrated with Outlet Fractionation for Dynamic Cell Culture.

Certain cell and tissue functions operate within the dynamic time scale of minutes to hours that are poorly resolved by conventional culture systems. This work has developed a low-cost perfusion bioreactor system that allows culture medium to be continuously perfused into a cell culture module and fractionated in a downstream module to measure dynamics on this scale. The system is constructed almost entirely from commercially available parts and can be parallelized to conduct independent experiments in conventional multi-well cell culture plates simultaneously. This video article demonstrates how to assemble the base setup, which requires only a single multichannel syringe pump and a modified fraction collector to perfuse up to six cultures in parallel. Useful variants on the modular design are also presented that allow for controlled stimulation dynamics, such as solute pulses or pharmacokinetic-like profiles. Importantly, as solute signals travel through the system, they are distorted due to solute dispersion. Furthermore, a method for measuring the residence time distributions (RTDs) of the components of the perfusion setup with a tracer using MATLAB is described. RTDs are useful to calculate how solute signals are distorted by the flow in the multi-compartment system. This system is highly robust and reproducible, so basic researchers can easily adopt it without the need for specialized fabrication facilities.

Synthesis and evaluation of 2-(18)F-fluoro-5-iodo-3-[2-(S)-3,4-dehydropyrrolinylmethoxy]pyridine ((18)F-Niofene) as a potential imaging agent for nicotinic α4ß2 receptors.

Nicotinic α4ß2 acetylcholine receptors (nAChRs) have been implicated in various pathophysiologies including neurodegenerative diseases. Currently, 2-(18)F-A85380 (2-FA) and 5-(123)I-A85380 (5-IA) are used separately in human PET and SPECT studies respectively and require >4-6 hours of scanning. We have developed 2-fluoro-5-iodo-3-[2-(S)-3-dehydropyrrolinylmethoxy]pyridine (niofene) as a potential PET/SPECT imaging agent for nAChRs with an aim to have rapid binding kinetics similar to that of (18)F-nifene used in PET studies. Niofene exhibited a 10-fold better in vitro binding affinity in rat brain than that of nicotine. The relative binding of niofene was similar to that of niodene and twice as better as that of nifene. In vitro autoradiography in rat brain slices alongside niodene indicated selective binding of niofene to regions consistent with α4ß2 receptor distribution. Niofene, 10 nM, displaced >70% of (3)H-cytisine bound to α4ß2 receptors in rat brain slices. Radiolabeling of (18)F-niofene was achieved in 10-15% radiochemical yield in high specific activities >2 Ci/µmol and showed rapid in vivo kinetics similar to that of (18)F-nifene and (18)F-nifrolene. In vivo PET in rats showed rapid uptake in the brain and selective localization in receptor regions such as the thalamus (TH). Pseudoequilibrium with (18)F-niofene was achieved in 30-40 minutes, which is similar to that of (18)F-nifene. Further evaluation of (18)F-niofene as a potential PET imaging agent is underway. Future studies will be conducted to radiolabel niofene with iodine-123 for use in SPECT imaging.

Collaborative prescribing: a qualitative exploration of a role for pharmacists in mental health.

BACKGROUND: Collaborative prescribing has been proposed as an extension of practice for advanced pharmacist practitioners. A lack of research investigating how pharmacists might be most effective as prescribers in mental health was identified. OBJECTIVE: To explore health professionals' and consumers' attitudes and beliefs that relate to the role of specialist mental health pharmacists working as collaborative prescribers within their advanced scope of practice in secondary care. METHODS: Semistructured interviews were conducted with key informants in the New Zealand mental health sector. Participants were selected via a purposive sampling method, including health professionals (n=9) and consumers (n=3). NVivo software was used to analyze data, using a thematic analysis approach to develop a series of key themes from the interviews. Common themes were extracted, which were used to gather results and draw conclusions. RESULTS: The key findings include a widespread acknowledgment of the role of specialist pharmacists as collaborative prescribers in mental health and as integral members of the multidisciplinary team; however, consumers were unaware of pharmacists' role in secondary care. The role was seen to extend current practice particularly in medication management after assessment and diagnosis by a medical practitioner. Concerns regarding demonstrating competence, practitioner role/boundary confusion, insufficient training and workforce development, hesitancy by pharmacists to extend role, consumer awareness, and public perception of the traditional pharmacist role were identified. Solutions discussed included education by the profession; relationship building, training, and robust competency assessments; and a structured framework for implementing a collaborative prescribing model. CONCLUSIONS: This study suggests there was recognition and acceptance of the role that specialist pharmacist practitioners could play in contributing to the care of mental health consumers as collaborative prescribers; their medication expertise being regarded highly. Further research is necessary to investigate how current resource constraints will allow for collaborative prescribing to be implemented within the context of mental health practice.

The development of spatial frequency discrimination.

We compared thresholds for discriminating spatial frequency for children aged 5, 7, and 9 years, and adults at two baseline spatial frequencies (1 and 3 cpd). In Experiment 1, the minimum change from baseline necessary to detect a change in spatial frequency from either baseline decreased with age from 34% in 5-year-olds to 11% in 7-year-olds, 8% in 9-year-olds, and 6% in adults. The data were best fit by an exponential function reflecting the rapid improvement in thresholds between 5 and 7 years of age and more gradual improvement thereafter (r(2) = 0.50, p < 0.0001). In Experiment 2, 5-year-olds' thresholds were higher than those of adults, even when memory demands were eliminated by presenting the two spatial frequencies side by side for an unlimited time. The pattern of development for sensitivity to spatial frequency (this study) resembles those for the development of sensitivity to orientation (T. L. Lewis, S. E. Chong, & D. Maurer, 2009) and contrast (D. Ellemberg, T. L. Lewis, C. H. Lui, & D. Maurer, 1999). The similar patterns are consistent with theories of common underlying mechanisms in primary visual cortex (A. Vincent & D. Regan, 1995; W. Zhu, M. Shelley, & R. Shapley, 2008) and suggest that those mechanisms continue to develop throughout childhood.