Surviving septic patients endotyped with a functional assay demonstrate active immune responses

Frontiers in Immunology, Journal Year: 2024, Volume and Issue: 15

Published: Oct. 14, 2024

Introduction Sepsis is a complex clinical syndrome characterized by heterogenous host immune response. Historically, static protein and transcriptomic metrics have been employed to describe the underlying biology. Here, we tested hypothesis that ex vivo functional TNF expression as well an immunologic endotype based on both IFNγ could be used model outcomes in sepsis patients. Methods This prospective, observational study of patient samples collected from SPIES consortium included patients at five health systems enrolled over 17 months, with 46 healthy control patients, 68 ICU without sepsis, 107 sepsis. Whole blood was day 1, 4, 7 admission. Outcomes in-hospital 180-day mortality non-favorable discharge disposition defined skilled nursing facility, long-term acute care or hospice. ELISpot assays were conducted quantify [stimulated lipopolysaccharide (LPS)] (stimulated anti-CD3/CD28 mAb), which then for assignment one four subgroups including ‘immunocompetent’, ‘immunosuppressed endotype’, two ‘mixed’ endotypes. Results spot-forming units significantly increased septic CINS days 4 compared subjects. In contrast, per cell lower critically ill non-septic (CINS) Early increases total associated favorable mortality. ‘Immunocompetent’ 1 had higher proportion discharges ‘immunosuppressed’ endotype. Similarly, ‘immunocompetent’ survival Finally, among decreased Conclusions Increased whole improved outcomes. Further, early survival. The ability functionally stratify function may allow identification future modulating therapies.

Language: Английский

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Temporal Changes in Innate and Adaptive Immunity During Sepsis as Determined by ELISpot

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Dec. 14, 2023

ABSTRACT Background The inability to evaluate host immunity in a rapid quantitative manner patients with sepsis has severely hampered development of novel immune therapies. ELISpot assay is functional bioassay that measures the number cytokine-secreting cells and relative amount cytokine produced at single-cell level. A key advantage its excellent dynamic range enabling more precise quantifiable assessment immunity. Herein, we tested hypothesis on whether can detect changes both innate adaptive as they often occur during sepsis. We also could effect drug therapies modulate Methods Mice were made septic using sublethal cecal ligation puncture (CLP). Blood spleens harvested serially ex vivo IFN-γ TNF-α production compared by ELISA. capability due therapy dexamethasone, IL-7, arginine was evaluated. Results confirmed decreased responsiveness progression. More importantly, able response immune-modulatory reagents, for example arginine, IL-7 readily manner, predicted reagents known mechanisms action. ELISA results tended parallel one another although some differences noted. Conclusion offers unique assess status over time. presented herein demonstrate be used follow effects drugs ameliorate sepsis-induced dysfunction. This would major advance guiding new

Language: Английский

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Development and optimization of a diluted whole blood ELISpot assay to test immune function

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Jan. 23, 2024

ABSTRACT Background Sepsis remains a leading cause of death worldwide with no proven immunomodulatory therapies. Stratifying Patient Immune Endotypes in (‘SPIES’) is prospective, multicenter observational study testing the utility ELISpot as functional bioassay specifically measuring cytokine-producing cells after stimulation to identify immunosuppressed endotype, predict clinical outcomes septic patients, and test potential immune stimulants for development. Most protocols call isolation PBMC prior their inclusion assay. In contrast, we developed diluted whole blood (DWB) protocol that has been validated across multiple laboratories. Methods Heparinized was collected from healthy donors patients tested under different conditions evaluate impact dilution, stimulant concentration, storage, length on ex vivo IFNγ TNFα production measured by ELISpot. Results We demonstrate dynamic range dilutions give robust cytokine response stimuli. Additionally, wide concentrations can be utilized induce production. Further modifications anticoagulated stored up 24 hours at room temperature without losing significant functionality. Finally, show brief 4 allowing substantial decrease processing time. Conclusions The data feasibility using measure capacity within DWB variety inform clinicians extent dysregulation patients.

Language: Английский

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Determining potential immunomodulatory drug efficacy in sepsis using ELISpot

Published: July 16, 2024

ABSTRACT Purpose This study evaluated the ability of ELISpot to identify immuno-modulatory drug therapies for their potential efficacy in patients with sepsis. Methods was performed using diluted whole blood from 61 septic and 48 healthy matched controls. Innate adaptive immunity were by ex vivo stimulated production TNF-α IFN-γ respectively. Potential determined drugs’ effects increase or decrease number cytokine-producing cells amount cytokine produced per cell as spot size intensity. The corticosteroid dexamethasone its down modulate production. TLR7/8 agonist resiquimod (R848) T-cell stimulants IL-7 anti-PD-1 mAb tested enhance immune responses Results Spontaneous varied among subjects patients. LPS stimulation increased total 1,648% 1,929% Conversely, diminished 71% 61% IL-7, but not markedly both (127%) (79%). Dexamethasone also reduced anti-CD3/CD28 54%; while ameliorated dexamethasone-induced suppression. significantly enhanced lymphocyte function over 90% Conclusion can reveal host response patterns drugs selectively down– up-regulate patient immunity. Furthermore, detect effect specific independently regulate innate could enable precision-based

Language: Английский

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