A Measure of the Impact on Real-Time Patient Care of Evidence-based Medicine Logs.

Introduction: Evidence-based medicine (EBM) is a critical skill for physicians, and EBM competency has been shown to increase implementation of best medical practices, reduce medical errors, and increase patient-centered care. Like any skill, EBM must be practiced, receiving iterative feedback to improve learners' comprehension. Having residents document patient interactions in logbooks to allow for residency program review, feedback, and documentation of competency has been previously described as a best practice within emergency medicine (EM) to document practice-based learning (PBL) competency. Quantifying how residents use the information they query, locate, evaluate, and apply while providing direct patient care can measure the efficacy of EBM education and provide insight into more efficient ways of providing medical care. Methods: Practice-based learning logs were surveys created to record resident EBM activity on-shift and were placed into our residency management software program. Residents were required to submit 3-5 surveys of EBM activity performed during a 28-day rotation during which additional information was sought. This study included all PBL logs completed by EM residents from June 1, 2013-May 11, 2020. Using qualitative methodology, a codebook was created to analyze residents' free-text responses to the prompt: "Based on your research, would you have done anything differently?" The codebook was designed to generate a three-digit code conveying the effect of the researched information on the patient about whom the log was written, as well as whether the information would affect future patient care and whether these decisions were based on scientific evidence. Results: A total of 10,574 logs were included for primary analysis. In total, 1,977 (18.7%) logs indicated that the evidence acquired through research would affect future patient care. Of these, 392 (3.7%) explicitly stated that the EBM activity conducted as part of our project led to real-time changes in patient care in the ED and would change future management of patients as well. Conclusion: We present a proof of concept that PBL log activity can lead to integration of evidence-based medicine into real-time patient care. While a convenience sample, our cohort recorded evidence of both lifelong learning and application to patient care.

Bispecific CD33/CD123 targeted chimeric antigen receptor T cells for the treatment of acute myeloid leukemia.

CD33 and CD123 are expressed on the surface of human acute myeloid leukemia blasts and other noncancerous tissues such as hematopoietic stem cells. On-target off-tumor toxicities may limit chimeric antigen receptor T cell therapies that target both CD33 and CD123. To overcome this limitation, we developed bispecific human CD33/CD123 chimeric antigen receptor (CAR) T cells with an "AND" logic gate. We produced novel CD33 and CD123 scFvs from monoclonal antibodies that bound CD33 and CD123 and activated T cells. Screening of CD33 and CD123 CAR T cells for cytotoxicity, cytokine production, and proliferation was performed, and we selected scFvs for CD33/CD123 bispecific CARs. The bispecific CARs split 4-1BB co-stimulation on one scFv and CD3ζ on the other. In vitro testing of cytokine secretion and cytotoxicity resulted in selecting bispecific CAR 1 construct for in vivo analysis. The CD33/CD123 bispecific CAR T cells were able to control acute myeloid leukemia (AML) in a xenograft AML mouse model similar to monospecific CD33 and CD123 CAR T cells while showing no on-target off-tumor effects. Based on our findings, human CD33/CD123 bispecific CAR T cells are a promising cell-based approach to prevent AML and support clinical investigation.

4-1BB and optimized CD28 co-stimulation enhances function of human mono-specific and bi-specific third-generation CAR T cells.

BACKGROUND: Co-stimulatory signals regulate the expansion, persistence, and function of chimeric antigen receptor (CAR) T cells. Most studies have focused on the co-stimulatory domains CD28 or 4-1BB. CAR T cell persistence is enhanced by 4-1BB co-stimulation leading to nuclear factor kappa B (NF-κB) signaling, while resistance to exhaustion is enhanced by mutations of the CD28 co-stimulatory domain. METHODS: We hypothesized that a third-generation CAR containing 4-1BB and CD28 with only PYAP signaling motif (mut06) would provide beneficial aspects of both. We designed CD19-specific CAR T cells with either 4-1BB or mut06 together with the combination of both and evaluated their immune-phenotype, cytokine secretion, real-time cytotoxic ability and polyfunctionality against CD19-expressing cells. We analyzed lymphocyte-specific protein tyrosine kinase (LCK) recruitment by the different constructs by immunoblotting. We further determined their ability to control growth of Raji cells in NOD scid gamma (NSG) mice. We also engineered bi-specific CARs against CD20/CD19 combining 4-1BB and mut06 and performed repeated in vitro antigenic stimulation experiments to evaluate their expansion, memory phenotype and phenotypic (PD1+CD39+) and functional exhaustion. Bi-specific CAR T cells were transferred into Raji or Nalm6-bearing mice to study their ability to eradicate CD20/CD19-expressing tumors. RESULTS: Co-stimulatory domains combining 4-1BB and mut06 confers CAR T cells with an increased central memory phenotype, expansion, and LCK recruitment to the CAR. This enhanced function was dependent on the positioning of the two co-stimulatory domains. A bi-specific CAR targeting CD20/CD19, incorporating 4-1BB and mut06 co-stimulation, showed enhanced antigen-dependent in vitro expansion with lower exhaustion-associated markers. Bi-specific CAR T cells exhibited improved in vivo antitumor activity with increased persistence and decreased exhaustion. CONCLUSION: These results demonstrate that co-stimulation combining 4-1BB with an optimized form of CD28 is a valid approach to optimize CAR T cell function. Cells with both mono-specific and bi-specific versions of this design showed enhanced in vitro and in vivo features such as expansion, persistence and resistance to exhaustion. Our observations validate the approach and justify clinical studies to test the efficacy and safety of this CAR in patients.

CD28 Costimulatory Domain-Targeted Mutations Enhance Chimeric Antigen Receptor T-cell Function.

An obstacle to the development of chimeric antigen receptor (CAR) T cells is the limited understanding of CAR T-cell biology and the mechanisms behind their antitumor activity. We and others have shown that CARs with a CD28 costimulatory domain drive high T-cell activation, which leads to exhaustion and shortened persistence. This work led us to hypothesize that by incorporating null mutations of CD28 subdomains (YMNM, PRRP, or PYAP), we could optimize CAR T-cell costimulation and enhance function. In vivo, we found that mice given CAR T cells with only a PYAP CD28 endodomain had a significant survival advantage, with 100% of mice alive after 62 days compared with 50% for mice with an unmutated endodomain. We observed that mutant CAR T cells remained more sensitive to antigen after ex vivo antigen and PD-L1 stimulation, as demonstrated by increased cytokine production. The mutant CAR T cells also had a reduction of exhaustion-related transcription factors and genes such as Nfatc1, Nr42a, and Pdcd1 Our results demonstrated that CAR T cells with a mutant CD28 endodomain have better survival and function. This work allows for the development of enhanced CAR T-cell therapies by optimizing CAR T-cell costimulation.

Organization and function of tension-dependent complexes at adherens junctions.

Adherens junctions provide attachments between neighboring epithelial cells and a physical link to the cytoskeleton, which enables them to sense and transmit forces and to initiate biomechanical signaling. Examination of the Ajuba LIM protein Jub in Drosophila embryos revealed that it is recruited to adherens junctions in tissues experiencing high levels of myosin activity, and that the pattern of Jub recruitment varies depending upon how tension is organized. In cells with high junctional myosin, Jub is recruited to puncta near intercellular vertices, which are distinct from Ena-containing puncta, but can overlap Vinc-containing puncta. We identify roles for Jub in modulating tension and cellular organization, which are shared with the cytohesin Step, and the cytohesin adapter Sstn, and show that Jub and Sstn together recruit Step to adherens junctions under tension. Our observations establish Jub as a reporter of tension experienced at adherens junctions, and identify distinct types of tension-dependent and tension-independent junctional complexes. They also identify a role for Jub in mediating a feedback loop that modulates the distribution of tension and cellular organization in epithelia.