Biology subjects

Kelly, T. K.

Publications and source records attributed to Kelly, T. K..

4 recordsLinked to original sources

bioRxiv2024

Understanding Complex Chromatin Dynamics of Primary Human Neutrophils During PMA Induced NETosis

BackgroundPrimary human neutrophils play a pivotal role in innate immunity, mainly through the formation of neutrophil extracellular traps (NETs) in a process known as NETosis. This cell-death pathway is crucial for combating infections but is also implicated in many inflammatory diseases such as sepsis, systemic lupus erythematosus, rheumatoid arthritis, and others. MethodsThe study presented here investigates chromatin dynamics during NETosis by stimulating primary human neutrophils with phorbol 12-myristate 13-acetate (PMA). We adapt the ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) method to isolated neutrophils and characterize a time-dependent chromatin response. ResultsWe find that chromatin accessibility patterns are consistent across individual donors and most chromatin changes occur within 30 minutes, with many continuing across the 90 minutes assessed in this study. Regulatory regions gaining accessibility are associated with activity of pathways that have been implicated in NOX-dependent NET formation. ConclusionsOur findings enhance the understanding of the chromatin changes underlying NETosis and also identify potential early-acting targets for modulating this process in inflammatory diseases.

immunology↗

bioRxiv2024

Long-read sequencing reveals aberrant fragmentation patterns and origins of circulating DNA in cancer

Circulating cell-free DNA (cfDNA) carries fragmentation patterns that serve as biomarkers of cancer, but standard sequencing approaches miss large portions of the fragment length spectrum. In addition to altered fragmentation patterns, cancer patients often have elevated levels of cfDNA, but the underlying mechanisms are not well understood. To address both questions, we analyzed cancer cases with elevated cfDNA levels using Oxford Nanopore (ONT) sequencing. Long-read ONT sequencing captures the full spectrum of cfDNA fragment lengths and enables cell type inference based on DNA methylation markers. One cohort included cases from several cancer types with elevated cfDNA levels, and a second consisted of patients from a single neuroendocrine cancer study. In each cohort, cases with the highest cfDNA levels showed either hypofragmentation (excess fragments of 1-4 kb) or hyperfragmentation (excess fragments <145 bp). Hypofragmentation reflected blood cell DNA released during delayed sample processing, bearing DNASE1L3-associated hallmarks, while in one cohort we also observed ultra-long fragments (>7.5 kb) lacking these hallmarks and consistent with plasma lysis. By contrast, hyperfragmented samples often had elevated levels of both cancer- and blood-derived DNA, indicating an inflammatory or other system process rather than cancer-specific origin. These findings clarify the distinction between biological and artifactual fragmentation, expand our understanding of cfDNA biology, and highlight long-read sequencing as a powerful tool for biomarker discovery.

cancer biology↗

bioRxiv2023

Novel rapid high-throughput method of NETosis Induction and Inhibition with physiological triggers and inhibitors

Neutrophils, the most abundant white blood cells in humans, play pivotal roles in innate immunity, rapidly migrating to sites of infection and inflammation to phagocytose, neutralize, and eliminate invading pathogens. Neutrophil Extracellular Trap (NET) formation in response to pathogens is increasingly recognized as an essential rapid innate immune response, but when dysregulated contributes to pathogenesis of sepsis and immunothrombotic disease. Current models of NETosis are limited, routinely employing non-physiological triggers that can bypass natural NET regulatory pathways. Models utilizing isolated neutrophils and immortalized cell lines, do not reflect the complex biology underlying neutrophil activation and NETosis, that occurs in whole-blood. Here we describe a novel, high-throughput ex-vivo whole blood induced NETosis model using combinatorial pooling of native NETosis inducing factors in a more biologically relevant Synthetic-Sepsis model. We found different combinations of factors evoked distinct neutrophil responses in the rate of NET generation and/or magnitude of NETosis. Despite inter-donor variability, similar sets of pro-inflammatory molecules induced consistent responses across donors. We found at least three biological triggers were necessary to induce NETosis in our system including either TNF- or LT-. To our knowledge, we report the first human ex-vivo model utilizing naturally occurring molecules to induce NETosis in whole blood. This approach could be used for drug screening and, importantly, inadvertent activators of NETosis. These findings emphasize the importance of investigating neutrophil physiology in a biologically relevant context to enable a better understanding of disease pathology, risk factors, and therapeutic targets, potentially, providing novel strategies for disease intervention and treatment. EssentialsO_LINETosis is a vital immune response, but dysregulation leads to disastrous health outcomes. C_LIO_LICurrent NETosis models dont reflect the complex endogenous signaling that occurs in whole blood C_LIO_LINetosis induction stimuli differs between isolated neutrophils and whole blood. C_LIO_LIA minimum of three physiological factors are required to induce NETosis in whole blood. C_LI

immunology↗

bioRxiv2023

Monitoring plasma nucleosome concentrations to measure of disease response and progression in dogs with hematopoietic malignancies.

BackgroundHematopoietic malignancies are extremely common in pet dogs and represent nearly 30% of the malignancies diagnosed in this population each year. Clinicians commonly use existing tools such as physical exam findings, radiographs, ultrasound and baseline blood work to monitor these patients for treatment response and remission. Circulating biomarkers, such as prostate specific antigen or carcinoembryonic antigen, can be useful tools for monitoring treatment response and remission status in human cancer patients. To date, there has a been a lack of useful circulating biomarkers available to veterinary oncology patients. MethodsCirculating plasma nucleosome concentrations were evaluated at diagnosis, throughout treatment and during remission monitoring for 40 dogs with lymphoma, acute myelogenous leukemia and multiple myeloma. Additionally, C-reactive protein and thymidine kinase-1 levels were recorded. ResultsPlasma nucleosome concentrations were significantly higher at diagnosis and progressive disease than they were when dogs were in remission. All but two dogs had plasma nucleosome concentrations that returned to the low range during treatment. These two dogs had the shortest progression free and overall survival times. Dogs with the highest plasma nucleosome concentrations had a significantly shorter first progression free survival than dogs with lower plasma nucleosome concentrations at diagnosis. Plasma nucleosome concentrations correlated better with disease response and progression than either thymidine kinase or C reactive protein. ConclusionsPlasma nucleosome concentrations can be a useful tool for treatment monitoring and disease progression in dogs with hematopoietic malignancies.

cancer biology↗