The discovery of induced pluripotent stem (iPS) cells, barely more than a decade ago, dramatically transformed the study of stem cells and introduced a completely new way to approach many human health concerns. Although advances have pushed the field forward, human application remains some years away, in part due to the need for an in-depth mechanistic understanding. The role of Wnts in stem cells predates the discovery of iPS cells with Wnts established as major pluripotency promoting factors. Most work to date has been done using mouse and tissue culture models and few attempts have been made in other model organisms, but the recent combination of clustered regularly interspaced short palindromic repeats (CRISPR) gene editing with iPS cell technology provides a perfect avenue for exploring iPS cells in model organisms. Drosophila is an ideal organism for such studies, but fly iPS cells have not yet been made. In this opinion article, we draw parallels between Wnt signaling in human and Drosophila stem cell systems, propose ways to obtain Drosophila iPS cells, and suggest ways to exploit the versatility of the Drosophila system for future stem cell studies.
During animal development, complex signals determine and organize a vast number of tissues using a very small number of signal transduction pathways. These developmental signaling pathways determine cell fates through a coordinated transcriptional response that remains poorly understood. The Wnt pathway is involved in a variety of these cellular functions, and its signals are transmitted in part through a β-catenin/TCF transcriptional complex. Here we report an in vivo Drosophila assay that can be used to distinguish between activation, de-repression and repression of transcriptional responses, separating upstream and downstream pathway activation and canonical/non-canonical Wnt signals in embryos. We find specific sets of genes downstream of both β-catenin and TCF with an additional group of genes regulated by Wnt, while the non-canonical Wnt4 regulates a separate cohort of genes. We correlate transcriptional changes with phenotypic outcomes of cell differentiation and embryo size, showing our model can be used to characterize developmental signaling compartmentalization in vivo.
Background Emergency Department (ED) airway registries are formalized methods to collect and document airway practices and outcomes. Airway registries have become increasingly common in EDs globally; yet there is no consensus of airway registry methodology or intended utility. This review builds on previous literature and aims to provide a thorough description of international ED airway registries and discuss how airway registry data is utilized. Methods A search of Medline, Embase, Scopus, Cochrane Libraries, Web of Science, and Google Scholar was performed with no date limitations applied. English language full-text publications and grey literature from centres implementing an ongoing airway registry to monitor intubations performed in mainly adult patients in an ED setting were included. Non-English publications and publications describing airway registries to monitor intubation practices in predominantly paediatric patients or settings outside of the ED were excluded. Study screening for eligibility was performed by two team members individually, with any disagreements resolved by a third team member. Data was charted using a standardized data charting tool created for this review. Results Our review identified 124 eligible studies from 22 airway registries with a global distribution. We found that airway registry data is used for quality assurance, quality improvement, and clinical research regarding intubation practices and contextual factors. This review also demonstrates that there is a great deal of heterogeneity in definitions of first-pass success and adverse events in the peri-intubation period. Conclusions Airway registries are used as a crucial tool to monitor and improve intubation performance and patient care. ED airway registries inform and document the efficacy of quality improvement initiatives to improve intubation performance in EDs globally. Standardized definitions of first-pass success and peri-intubation adverse events, such as hypotension and hypoxia, may allow for airway management performance to be compared on a more equivalent basis and allow for the development of more reliable international benchmarks for first-pass success and rates of adverse events in the future.
Acute lymphoblastic leukemia (ALL) is a disease of the bone marrow (BM) microenvironment, in which ALL-stroma interactions, signaling molecules, and low oxygen tension all contribute to leukemia development and maintenance. The cytokine interleukin 12 (IL12) is a key primer of the initial specific T-cell immune response for resisting cancer progression, however its potential as an anti-leukemia immunotherapeutic agent has been largely under-examined. One potential reason for this oversight is the inability of current experimental systems to recapitulate the complex BM microenvironmental system ex vivo or obtain the resolution necessary in vivo to study the precise mechanism of effect for such proposed immunotherapies. Recently, our lab has developed a novel femur window chamber (FWC) intravital imaging mouse model capable of long-term spatiotemporal assessment of leukemias within the in vivo femoral BM at cellular resolution. We used this technique to characterize a new cell-based IL12 immunotherapy, in which patients are injected with their own blast cells engineered to express IL12. This approach has shown significant potential in preliminary preclinical trials, although its mechanism of effect is still unknown. Our study sought to establish the precise interaction between the modified IL12-expressing blast cells with the BM ALL population and immune system to determine a) how long post-ALL engraftment IL12 therapy continue to be effective, b) the optimal treatment administration route, and c) whether repeated infusions will yield added benefit. The murine lymphoblastic cell line, 70Z/3, was transfected with either a constitutively active GFP marker (70Z/3-GFP) or a lentivirus expressing mCherry and IL12 (70Z/3-IL12-mCherry). The FWC was installed by shaving the femoral corticalis to reveal the BM cavity and securing a cover glass on top. Mice were then intravenously (IV) injected with 3x106 70Z/3-GFP cells 2 weeks post-surgery. Fluorescent cellular activity within the BM was monitored two times a week for four weeks via confocal microscopy (Zeiss LSM710). 3x106 70Z/3-IL12-mCherry cells were then injected into mice IV, intraperitoneally (IP), or intrafemorally (IF) at each experimental time point following initial inoculation. Leukemic burden was quantified as the percent total GFP signal within the BM space while immune cell activity was monitored using injected fluorescently-conjugated antibodies and ex vivo flow cytometry. Nonfluorescent leukemic and healthy mice were also imaged as controls. Intravital imaging revealed that IV injected 70Z/3-GFP cells first enter the BM, on average, 10 days post-inoculation and achieve 90% infiltration within 3 weeks. Based on this timeline, we evaluated the effect of IL12 treatment administered 1, 2, or 3 weeks post-inoculation, as well as at the time of inoculation itself (co-injection). Mice treated with a single administration of 70Z/3-IL12-mCherry cells at 1 week showed a 26% reduction (p<0.05) in overall leukemia burden at the experimental endpoint, while mice treated at 3 weeks did not have a statistically significantly reduction in burden compared to untreated controls. We also observed a significant decline in BM immune cell populations, particularly macrophages, associated with increasing ALL percentage. We hypothesize that IL12 therapy is only effective below a specific threshold of disease, which corresponds to a minimum effective immune population still remaining in the BM. Current efforts are now underway to characterize this threshold and test the ability of repeated infusions to maintain immune cells within this effective range for the entirety of the treatment course. Interestingly, 70Z/3-IL12-mCherry cells co-injected with 70Z/3-GFP cells prevented ALL establishment in the BM altogether. Additionally, while IP injection has been the sole treatment administration technique used in previous IL12 studies, our results indicate that IV administration is the more effective option; not only do IV injected cells reach the BM faster and with greater quantity as compared to all other techniques tested, but it is also the more clinically relevant modality. Our study not only demonstrates the ability of cell-based IL12 therapy to elicit a therapeutic response in ALL-bearing mice, it also reveals important insights into the biology of the immunotherapy, which can be applied to further augment the treatment's effect. Disclosures DaCosta: SBI ALApharma Canada: Current Employment, Current equity holder in private company; MolecuLight Inc.: Current Employment, Current equity holder in private company, Patents & Royalties: Device and Method for Fluorescence-Based Imaging and Monitoring.
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