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Background The objective of this work to calculate prescribed quantity of an active pharmaceutical ingredient (API) in prescription medications for human use, to facilitate research on the prediction of amount of API released to the environment and create an open-data tool to facilitate spatiotemporal and long-term prescription trends for wider usage. Design We have developed an R package, PrAna to calculate the prescribed quantity (in kg) of an APIs by postcode using England’s national level prescription data provided by National Health Service, for the years 2015–2018. Datasets generated using PrAna can be visualized in a real-time interactive web-based tool, PrAnaViz to explore spatiotemporal and long-term trends. The visualisations can be customised by selecting month, year, API, and region. Results PrAnaViz’s targeted API approach is demonstrated with the visualisation of prescribed quantities of 14 APIs in the Bath and North East Somerset (BANES) region during 2018. Once the APIs list is loaded, the back end retrieves relevant data and populates the graphs based on user-defined data features in real-time. These plots include the prescribed quantity of APIs over a year, by month, and individual API by month, general practice, postcode, and medicinal form. The non-targeted API approach is demonstrated with the visualisation of clarithromycin prescribed quantities at different postcodes in the BANES region. Conclusion PrAna and PrAnaViz enables the analysis of spatio-temporal and long-term trends with prescribed quantities of different APIs by postcode. This can be used as a support tool for policymakers, academics and researchers in public healthcare, and environmental scientist to monitor different group of pharmaceuticals emitted to the environment and for prospective risk assessment of pharmaceuticals in the environment.

Lung cancer is a leading cause of death worldwide, with only a fraction of patients responding to immunotherapy. The correlation between increased T-cell infiltration and positive patient outcomes has motivated the search for therapeutics promoting T-cell infiltration. While transwell and spheroid platforms have been employed, these models lack flow and endothelial barriers, and cannot faithfully model T-cell adhesion, extravasation and migration through 3D tissue. Presented here is a 3D chemotaxis assay, in a lung tumor on chip model with 3D endothelium (LToC-Endo), to address this need. The described assay consists of a vascular tubule cultured under rocking flow, through which T-cells are added; a collagenous stromal barrier, through which T-cells infiltrate; and a chemoattractant/tumor compartment. Here, activated T-cells extravasate and infiltrate in response to gradients of rhCXCL11 and rhCXCL12. Adopting a T-cell activation protocol with a rest period enables proliferative burst prior to introducing T-cells into chips, increases T-cell expression of CXCR3 and CXCR4 receptors, and enhances assay sensitivity. In addition, incorporating this rest recovers endothelial activation in response to rhCXCL12. As a final control, we show that blocking ICAM-1 interferes with T-cell adhesion and chemotaxis. This microphysiological system, which mimics in vivo stromal and vascular barriers, can be used to evaluate potentiation of immune chemotaxis into tumors while probing for vascular responses to potential therapeutics. Finally, we propose a translational strategy by which this assay could be linked to preclinical and clinical models to support human dose prediction, personalized medicine, and the reduction, refinement, and replacement of animal models.

Inducible T cell Co-Stimulator (ICOS, CD278) is a receptor belonging to the CD28/CTLA-4 superfamily that is predominantly expressed on T cells following T cell receptor (TCR) activation and plays an important role in regulating adaptive immune response. ICOS represents possible node of therapeutic intervention for cancer, and as such, is under clinical evaluation. Although ICOS agonists have been shown to promote significant monotherapeutic efficacy in non-clinical studies, most patients do not respond to the single-agent approaches. Therefore, it is crucial to better understand the potential mechanisms of action as well as to develop novel combination strategies and biomarkers to best position therapeutic in the clinic. In an effort to identify novel combination partners and/or biomarkers for ICOS agonist, we utilized pooled in vivo genetic screening via CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 genome editing in the syngeneic CT26 colorectal mouse tumor model alongside agonist treatment for the target. We evaluated 509 druggable genes with four sgRNAs targeting each gene to identify genes where loss in CT26 tumor cells can increase sensitivity or resistance to this treatment. With a false discovery rate (FDR) Citation Format: Qila Sa, Hongyu Liang, Rumen Kostadinov, Jong Yu, Jeremy Waight, Stephanie Van Horn, Qing Xie, Sue Griffin, Chris Hopson, Ning Sun. In vivo CRISPR screening in syngeneic tumor models to identify novel combinations for cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr A18.

Only a subset of patients durable clinical responses to aPD-1 and/or aCTLA-4 immunotherapies, thus, developing new therapeutic agents to increase the proportion of responding patients is a priority. Combining aPD-1 with aLAG-3 has shown promising results; however, lack of mechanistic understanding of aPD-1/aLAG-3 synergy remains a barrier for its optimal clinical use. Here, we examined the mechanism of aPD-1/aLAG-3 synergy in multiple mouse models using flow cytometry and single cell RNA sequencing. Combined aPD-1/aLAG-3 immunotherapy significantly improved the survival of CT26 (BALB/c; colon carcinoma) and MCA-205 (C57BL/6; sarcoma) tumor-bearing mice compared to monotherapy. Regulatory T cells (Tregs) suppressed response to this therapy, as in the absence of CD4+ T cells, 100% of mice responded. To understand how responders overcome Treg suppression, we performed an in-depth analysis of tumor-infiltrating lymphocytes (TIL) comparing mice that responded to treatment (decreased tumor size post-treatment) to non-responders (same tumor growth trajectory as control). Responders had reduced Foxp3+ CD4+ Tregs in comparison to non-responders and, in addition, those Tregs had a ‘fragile’ phenotype, including a pro-inflammatory cytokine profile (TNF-a; IFN-g), increased LAG-3, and decreased NRP1 expression. Within responders, CD8+ TIL exhibited increased frequency, effector cytokine production (TNF-a; IFN-g), and LAG-3 expression as compared to non-responders. Together, these data suggest that aPD-1/aLAG-3 can reduce Treg frequency and function leading to expansion of active tumor-specific CD8+ T cells capable of supporting tumor regression and improved survival. Supported by the Providence Portland Medical Foundation and GlaxoSmithKline

To explore whether the variation in clinical response to immune checkpoint blockade (ICB) reflects intrinsic characteristics of tumor-infiltrating lymphocytes (TIL), TILs from multiple tumor types were analyzed by multiparameter flow cytometry. Recent work identified CD39+CD103+ double positive (DP) CD8 TIL as tumor-reactive, therefore we assessed this phenotype in primary NSCLC (n=28), BCa (n=23), HNSCC (n=23), and RCC (n=23) specimens. TILs from NSCLC and HNSCC had significantly higher frequencies of DP T cells than BCa and RCC (NSCLC: median=34.2, IQR=15.0–53.8%; HNSCC: median=28.6%, IQR=13.5–44.5%), while TILs from BCa and RCC had low frequencies of DP T cells (BCa: median=2.4%, IQR=1.1–3.9%; RCC: median=4.2%, IQR=1.5–16.6%). Additionally, DP cells in NSCLC and HNSCC were co-expressed immune checkpoint markers PD-1, TIM-3, and LAG-3 at a higher frequency than RCC or BCa (P Supported by GlaxoSmithKline and the Providence Portland Medical Foundation

Substantial evidence exists that social circumstances can affect children’s language development. As a result many children in socially deprived areas start school with delayed language, which may persist and adversely affect their attainment. We assessed the language of children in seven reception classes in a London (UK) borough and followed the progress of children with English as their first language (E1L) and with English as an additional language (EAL) during their first 2 years at school. Significant differences were found between schools. The effect of social factors on performance was reflected in a high correlation between the mean language score for each school and the percentage of children in the school receiving the pupil premium. Many of the children with EAL had very low scores reflecting their limited exposure to English prior to starting school. Most of these children attended schools where children with E1L also had low scores increasing the demands on the schools and their teachers. Children who had low initial scores made modest but significant progress during their reception year but failed to improve further during year 1 despite having non-verbal ability appropriate for their age. These results support previous findings that social deprivation can seriously delay language development, and that many children start school with weak communication skills. They add to previous findings by showing that the level of delay may differ substantially across schools in the same borough, by reporting data on children with EAL and by showing that children struggle to improve their abilities in the first 2 years of school.