Biology subjects

Kort, E. J.

Publications and source records attributed to Kort, E. J..

4 recordsLinked to original sources

bioRxiv2021

Identification and Characterization of Human Activation-Induced ChAT+CD4+ T Cells

Vasodilation is a cornerstone of inflammation physiology. By regulating vasodilation and tissue entry of T cells, CD4+ T lymphocytes expressing choline acetyltransferase (ChAT), a key enzyme for biosynthesis of the vasorelaxant acetylcholine (ACh), critically link immunity with vascular biology in mice. However, the characterization of primary human ChAT+ T cells remained elusive. Here, we identified human ChAT+ T cells and report that ChAT mRNA was induced by activation. Functional studies demonstrated that T cell-derived ACh increased muscarinic ACh-receptor dependent NO-synthase activity and vasorelaxation. Further, single-cell RNA-sequencing revealed ChAT+CD4+ T cells in blood from patients with severe circulatory failure and a high relative frequency of ChAT+CD4+ T cells correlated with better 30-day survival in this cohort. Our findings provide the first insights into ChAT biology in primary human T cells, linking ChAT+ T cells with vasorelaxation as well as survival in a cohort of critically ill patients.

immunology↗

bioRxiv2019

Toxicogenomic identification of repositioned therapy for a monogenic disease

The cost of drug development from initial concept to FDA approval has been estimated to be about 2.6 billion USD.1 This cost precludes development of targeted therapies for rare diseases such as monogenetic cardiomyopathies. As part of the Library of Integrated Network-based Cellular Signatures (LINCS) program funded by the NIH, the Broad Institute of MIT has publicly released transcriptional profiles quantifying the effects of more than 25,000 perturbagens on the expression of 978 genes in up to 77 cell lines.2 Transcriptomics has been shown to be a powerful tool in repurposing drugs3,4 and this dataset affords us the unique opportunity to systematically identify small molecule mimics or inhibitors of specific genes, thereby identifying novel treatments for genetic disorders. In this report, we take this approach to identify a novel drug therapy for a monogenic form of familial dilated cardiomyopathy with the transcriptional profile of FDA approved drugs. This approach could potentially be replicated for a wide range of monogenic diseases.

genomics↗

bioRxiv2019

Single cell transcriptomics identifies immunologic priming related to extra corporeal life support survival.

BackgroundDespite being a lifesaving intervention for the most critically ill and circulatory compromised patients, veno-arterial extra-corporeal life support (VA-ECLS) is associated with a mortality rate of nearly 60%. Understanding how the immune response to VA-ECLS either promotes or impedes survival would both enhance risk stratification and uncover new therapeutic strategies for these patients. However, conventional enumeration of peripheral blood mononuclear cells (PBMCs) and their subsets have failed to identify determinants of outcome among these cells. MethodsFlow cytometry and plasma cytokine measurement was combined with single cell RNASeq analysis of PBMCs from patients in circulatory shock being started on VA-ECLS to identify clinical, laboratory, and cellular features associated with 72 hour survival. ResultsNon-surviving patients exhibited higher plasma levels of the tissue aggressive inflammatory cytokines IL-1, IL-6, IL-12 and TNF-. Distribution of cells between conventional PBMC subtypes was not predictive of survival. Single cell RNASeq analysis of discriminatory markers within each PBMC subtype revealed that the proportion of CD8+ Natural Killer T-cells (NKT) that expressed CD52, a known immune-modulator, was associated with improved survival. This cell population correlated inversely with IL-6 production. CD8+/CD52+ NKT cells were quantified by flow cytometry in a second, validation cohort. Those patients with a high proportion of CD52+ cells among all CD8+ NKT cells had more severe disease relative to the low CD52+ group, but nevertheless were nearly 5 time less likely to die in the first 72 hours of VA-ECLS (p=0.043 by log rank test). ConclusionsCD8+/CD52+ NKT cells are associated with survival in patients undergoing VA-ECLS. Fluidics based scRNASeq can reveal important aspects of pathophysiology in complex disease states such as circulatory collapse and VA-ECLS. Further studies in animal models will be required to determine if stimulation of CD8+/CD52+ NKT cell expansion may be an effective therapeutic strategy in this patient population.

genomics↗

bioRxiv2019

Isolation of cardiomyocytes undergoing mitosis with complete cytokinesis

Rationale-Adult human cardiomyocytes (CMs) do not complete cytokinesis despite passing through the S-phase of the cell cycle. As a result polyploidization and multinucleation occur. In order to get a deeper understanding of the mechanisms surrounding division of CMs there is a crucial need for a technique to isolate CMs that complete cell division/cytokinesis. Objective-Markers of cell cycle progression based on DNA content cannot distinguish between mitotic CMs that fail to complete cytokinesis from those cells that undergo true cell division. With the use of molecular beacons (MB) targeting specific mRNAs we aimed to identify truly proliferative CMs derived from hiPSCs. Methods and Results-Fluorescence activated cell-sorting combined with molecular beacons was performed to sort CM populations enriched for mitotic cells. Expressions of cell-cycle specific genes were confirmed by means of RT-qPCR, single-cell RNA sequencing (scRNA-seq). We further characterized the sorted groups by proliferation assays and time-lapse microscopy which confirmed the proliferative advantage of MB-positive cell populations relative to MB-negative and G2/M populations. Gene expression analysis revealed that the MB-positive CM subpopulation exhibited patterns consistent with the biological processes of nuclear division, chromosome segregation, and transition from M to G1 phase. The use of dual-MBs targeting CDC20 and SPG20 mRNAs (CDC20+SPG20+) enabled the enrichment of cytokinetic events. Interestingly, cells that did not complete cytokinesis and remained binucleated were found to be CDC20-SPG20+ while polyploid CMs that replicated DNA but failed to complete karyokinesis were found to be CDC20-SPG20-. Conclusions-This study demonstrates a novel alternative to existing DNA content-based approaches for sorting CMs with true mitotic potential that can be used to study in detail the unique dynamics of CM nuclei during mitosis. Together with high-throughput scRNA-seq, our technique for sorting live CMs undergoing cytokinesis would provide a basis for future studies to uncover mechanisms underlying the development and regeneration of heart tissue.

cell biology↗