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Background As circulating DNA (cirDNA) is mainly detected as mononucleosome-associated circulating DNA (mono-N cirDNA) in blood, apoptosis has until now been considered as the main source of cirDNA. The mechanism of cirDNA release into the circulation, however, is still not fully understood. This work addresses that knowledge gap, working from the postulate that neutrophil extracellular traps (NET) may be a source of cirDNA, and by investigating whether NET may directly produce mono-N cirDNA. Methods We studied (1) the in vitro kinetics of cell derived genomic high molecular weight (gHMW) DNA degradation in serum; (2) the production of extracellular DNA and NET markers such as neutrophil elastase (NE) and myeloperoxidase (MPO) by ex vivo activated neutrophils; and (3) the in vitro NET degradation in serum; for this, we exploited the synergistic analytical information provided by specifically quantifying DNA by qPCR, and used shallow WGS and capillary electrophoresis to perform fragment size analysis. We also performed an in vivo study in knockout mice, and an in vitro study of gHMW DNA degradation, to elucidate the role of NE and MPO in effecting DNA degradation and fragmentation. We then compared the NET-associated markers and fragmentation size profiles of cirDNA in plasma obtained from patients with inflammatory diseases found to be associated with NET formation and high levels of cirDNA (COVID-19, N  = 28; systemic lupus erythematosus, N  = 10; metastatic colorectal cancer, N  = 10; and from healthy individuals, N  = 114). Results Our studies reveal that gHMW DNA degradation in serum results in the accumulation of mono-N DNA (81.3% of the remaining DNA following 24 h incubation in serum corresponded to mono-N DNA); “ex vivo” NET formation, as demonstrated by a concurrent 5-, 5-, and 35-fold increase of NE, MPO, and cell-free DNA (cfDNA) concentration in PMA-activated neutrophil culture supernatant, leads to the release of high molecular weight DNA that degrades down to mono-N in serum; NET mainly in the form of gHMW DNA generate mono-N cirDNA (2 and 41% of the remaining DNA after 2 h in serum corresponded to 1–10 kbp fragments and mono-N, respectively) independent of any cellular process when degraded in serum; NE and MPO may contribute synergistically to NET autocatabolism, resulting in a 25-fold decrease in total DNA concentration and a DNA fragment size profile similar to that observed from cirDNA following 8 h incubation with both NE and MPO; the cirDNA size profile of NE KO mice significantly differed from that of the WT, suggesting NE involvement in DNA degradation; and a significant increase in the levels of NE, MPO, and cirDNA was detected in plasma samples from lupus, COVID-19, and mCRC, showing a high correlation with these inflammatory diseases, while no correlation of NE and MPO with cirDNA was found in HI. Conclusions Our work describes the mechanisms by which NET and cirDNA are linked. In doing so, we demonstrate that NET are a major source of mono-N cirDNA independent of apoptosis and establish a new paradigm of the mechanisms of cirDNA release in normal and pathological conditions. We also demonstrate a link between immune response and cirDNA.

Circulating cell-free DNA (cfDNA) has been investigated as a screening tool for many diseases. To avoid expensive and time-consuming DNA isolation, direct quantification PCR assays can be established. However, rigorous validation is required to provide reliable data in the clinical and non-clinical context. Considering the International Organization for Standardization, as well as bioanalytical method validation guidelines, we provide a comprehensive procedure to validate assays for cfDNA quantification from blood plasma without DNA isolation. A 90 and 222 bp assay was validated to study the kinetics of cfDNA after exercise in patients with systemic lupus erythematosus (SLE). The assays showed ultra-low limit of quantification (LOQ) with 0.47 and 0.69 ng/ml, repeatability ≤ 11.6% (95% CI 8.1–20.3), and intermediate precision ≤ 12.1% (95% CI 9.2–17.7). Incurred sample reanalysis confirmed the precision of the procedure. The additional consideration of pre-analytical factors shows that centrifugation speed and temperature do not change cfDNA concentrations. In SLE patients cfDNA increases ~ twofold after a walking exercise, normalizing after 60 min of rest. The established assays allow reliable and cost-efficient quantification of cfDNA in minute amounts of plasma in the clinical setting. Additionally, the assay can be used as a tool to determine the impact of pre-analytical factors and validate cfDNA quantity and quality of isolated samples.

Background Cell-free DNA (cfDNA) has emerged as a relevant biomarker reflecting disease severity in hospitalised COVID-19 patients, correlating with respiratory failure and mortality. However, its utility has not yet been evaluated in general practitioner setting. Methods A prospective single-centre, two-arm, parallel, longitudinal cohort study conducted in a German general practice with four doctors between 8/2021 and 4/2022. Participants : Sixty-one outpatients with flu-like symptoms participated: 31 (10 men, 21 women) tested SARS-CoV-2 positive (COVID group); 30 (12 men, 18 women) were controls (control group). The groups were demographically similar. Primary outcome measures : Comparison of cfDNA levels between groups at day 0, 7 and 14. Secondary outcome measures : Correlations between cfDNA levels and laboratory and clinical parameters like blood counts, respiratory rate and oxygen saturation. Results cfDNA levels did not differ significantly between groups (F [1, 59] = 1.538, p  = 0.22): day 0: mean (± standard deviation) = 14.45 (± 6.24) ng/ml (COVID group) vs. 11.32 (± 4.79) ng/ml (control group); day 7: 14.46 (± 6.57) ng/ml vs. 12.53 (± 6.67) ng/ml; day 14: 12.94 (± 6.66) ng/ml vs. 12.93 (± 7.02) ng/ml. However, at t0, the integrity index was significantly lower in the COVID group (t0: 0.30 [±- 0.15] vs. 0.41 [± 0.1]; p  = 0.0127) increasing at t1 (0.38 [± 0.17]; p  = 0.008) and at t2 (0.42 [± 0.22]; p  < 0.001). Conclusion Unlike hospitalised patients, cfDNA levels did not differ significantly between outpatient groups. Therefore, a decision on the need for hospitalisation based on clinical and serological factors is still required. The significantly lower integrity index of the SARS-CoV-2 infected individuals indicates that their DNA kinetics differ from those of individuals infected with other respiratory pathogens. Trial registration : German Clinical Trials Register: DRKS00024722, Registration date: 10 March 2021.

Hyperinflammation and extensive cell damage characterize both COVID-19-sepsis and bacterial sepsis, contributing to poor clinical outcomes. Cell-free DNA (cfDNA), a damage-associated molecular pattern (DAMP), reflects ongoing tissue injury and may predict mortality. We aimed to evaluate cfDNA as a prognostic biomarker for 30-day mortality in ICU patients with COVID-19- vs. bacterial sepsis, and its association with inflammatory markers and disease progression. In a prospective observational study (ethics approval: 2020–15,535; DRKS-ID: DRKS00025222), cfDNA was quantified in 64 ICU patients (COVID-19-sepsis n = 27, bacterial sepsis n = 37) at four time points using quantitative PCR targeting 90 bp and 222 bp fragments of LINE-1 elements. An Integrity Index (222/90 bp) was calculated to infer the predominant mode of cell death. Nineteen healthy individuals served as controls. Associations with mortality and clinical parameters were analyzed using adjusted Cox regression, time-dependent models, and correlation analyses. Higher cfDNA levels (90 bp) within the first 24 h were strongly associated with 30-day ( p  = 0.003) and 180-day mortality ( p  = 0.003) in COVID-19-sepsis, but not in bacterial sepsis. COVID-19 patients showed significantly higher cfDNA levels ( p  < 0.01), which correlated with CRP, PCT, LDH, and lactate. The Integrity Index increased over time in bacterial sepsis and remained stable in COVID-19-sepsis, but was not predictive of survival. Elevated cfDNA levels were associated with ECMO therapy but not with renal replacement therapy. cfDNA is a valuable early prognostic biomarker in COVID-19-sepsis. Its rapid dynamics and strong correlation with clinical outcomes highlight its potential for real-time monitoring and risk stratification in viral sepsis.

Circulating cell‐free DNA (cfDNA) is a promising biomarker for physiological stress, including exercise‐induced responses. However, the lack of standardization in blood collection tubes (BCTs) for quantification of cfDNA hampers inter‐study comparisons. In this study, we assessed the impact of different BCTs on exercise‐induced cfDNA dynamics. Eleven participants [25 (SD 2.3) years of age] performed three different treadmill exercise protocols, including an all‐out test and combinations of constant and interval load. Blood samples were collected before, 5 min and 30 min post‐exercise using EDTA, lithium–heparin (LH) and serum BCTs. Concentrations of cfDNA were quantified using quantitative PCR. The cfDNA increased significantly across all protocols and BCTs. A significant effect of BCT on cfDNA concentrations (P = 0.034) was found, with serum showing higher concentrations than EDTA and LH. Although absolute differences from pre‐ to post‐exercise were comparable across BCTs (P = 0.476), fold changes differed significantly (P = 0.012), with the highest observed in EDTA and the lowest in serum. Bland–Altman analyses demonstrated better agreement between EDTA and LH compared with serum. Significant correlations of cfDNA with energy expenditure and peak oxygen uptake were found. These correlations were stronger in EDTA and LH than in serum. Our findings highlight the crucial influence of BCT choice on cfDNA measurements in exercise settings. Given that EDTA and LH reflected exercise load better, they could be preferred for exercise physiology research. This work underscores the need to account for the choice of BCT to improve data comparability across studies. Additionally, these findings might have broader implications for clinical settings where cfDNA is used as a biomarker. What is the central question of this study? This work examines how different blood collection tubes influence cell‐free DNA concentrations in standardized exercise settings. Additionally, correlations of exercise‐induced cell‐free DNA with exercise‐related parameters were investigated. What is the main finding and its importance? All blood collection tubes used (EDTA, lithium–heparin and serum) show typical exercise‐induced increases in cell‐free DNA. Fold changes and absolute concentrations differ between blood collection tubes, but absolute pre to post differences are comparable. EDTA and lithium–heparin show better agreement than serum with either EDTA or lithium–heparin. In addition, stronger correlations with exercise‐related parameters were observed.

Background Performing multiple high-intensity interval training (HIIT) sessions in a compressed period of time (approximately 7–14 days) is called a HIIT shock microcycle (SM) and promises a rapid increase in endurance performance. However, the efficacy of HIIT-SM, as well as knowledge about optimal training volumes during a SM in the endurance-trained population have not been adequately investigated. This study aims to examine the effects of two different types of HIIT-SM (with or without additional low-intensity training (LIT)) compared to a control group (CG) on key endurance performance variables. Moreover, participants are closely monitored for stress, fatigue, recovery, and sleep before, during and after the intervention using innovative biomarkers, questionnaires, and wearable devices. Methods This is a study protocol of a randomized controlled trial that includes the results of a pilot participant. Thirty-six endurance trained athletes will be recruited and randomly assigned to either a HIIT-SM (HSM) group, HIIT-SM with additional LIT (HSM + LIT) group or a CG. All participants will be monitored before (9 days), during (7 days), and after (14 days) a 7-day intervention, for a total of 30 days. Participants in both intervention groups will complete 10 HIIT sessions over 7 consecutive days, with an additional 30 min of LIT in the HSM + LIT group. HIIT sessions consist of aerobic HIIT, i.e., 5 × 4 min at 90–95% of maximal heart rate interspersed by recovery periods of 2.5 min. To determine the effects of the intervention, physiological exercise testing, and a 5 km time trial will be conducted before and after the intervention. Results The feasibility study indicates good adherence and performance improvement of the pilot participant. Load monitoring tools, i.e., biomarkers and questionnaires showed increased values during the intervention period, indicating sensitive variables. Conclusion This study will be the first to examine the effects of different total training volumes of HIIT-SM, especially the combination of LIT and HIIT in the HSM + LIT group. In addition, different assessments to monitor the athletes' load during such an exhaustive training period will allow the identification of load monitoring tools such as innovative biomarkers, questionnaires, and wearable technology. Trial Registration : clinicaltrials.gov, NCT05067426. Registered 05 October 2021—Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT05067426 . Protocol Version Issue date: 1 Dec 2021. Original protocol. Authors: TLS, NH.

Although it is widely accepted that cancer-derived extracellular vesicles (EVs) carry DNA cargo, the association of cell-free circulating DNA (cfDNA) and EVs in plasma of healthy humans remains elusive. Using a physiological exercise model, where EVs and cfDNA are synchronously released, we aimed to characterize the kinetics and localization of DNA associated with EVs. EVs were separated from human plasma using size exclusion chromatography or immuno-affinity capture for CD9+, CD63+, and CD81+ EVs. DNA was quantified with an ultra-sensitive qPCR assay targeting repetitive LINE elements, with or without DNase digestion. This model shows that a minute part of circulating cell-free DNA is associated with EVs. During rest and following exercise, only 0.12% of the total cfDNA occurs in association with CD9+/CD63+/CD81+EVs. DNase digestion experiments indicate that the largest part of EV associated DNA is sensitive to DNase digestion and only ~20% are protected within the lumen of the separated EVs. A single bout of running or cycling exercise increases the levels of EVs, cfDNA, and EV-associated DNA. While EV surface DNA is increasing, DNAse-resistant DNA remains at resting levels, indicating that EVs released during exercise (ExerVs) do not contain DNA. Consequently, DNA is largely associated with the outer surface of circulating EVs. ExerVs recruit cfDNA to their corona, but do not carry DNA in their lumen.

Physical activity impacts immune homeostasis and leads to rapid and marked increase in cell-free DNA (cfDNA). However, the origin of cfDNA during exercise remains elusive and it is unknown if physical activity could improve or interfere with methylation based liquid biopsy. We analyzed the methylation levels of four validated CpGs representing cfDNA from granulocytes, lymphocytes, monocytes, and non-hematopoietic cells, in healthy individuals in response to exercise, and in patients with hematological malignancies under resting conditions. The analysis revealed that physical activity almost exclusively triggered DNA release from granulocytes, highlighting the relevance as a pre-analytical variable which could compromise diagnostic accuracy. Graphical Abstract

Background This study aimed to evaluate acute and chronic exercise-induced changes in cell-free DNA (cfDNA) concentrations during a 7-day high-intensity interval training (HIIT) shock microcycle in trained endurance athletes. Thirty-five participants were randomly assigned to one of three groups: a HIIT-only group (HSM), a HIIT plus low-intensity training group (HSM + LIT), and a control group maintaining regular training. The intervention included 10 HIIT sessions (5 × 4 min at 90–95% maximum heart rate) over 7 days, with HSM + LIT completing an additional 30 min of low-intensity training after each session. Physiological exercise testing (PET) was conducted at baseline, 3-, 7-, and 14-days post-intervention. On days 2 and 7 during the intervention, HIIT sessions were supervised in both morning and afternoon, and venous blood samples were collected at rest, immediately post-exercise, and 30 min post-exercise to measure cfDNA for 90 and 222 bp fragments. Correlations between cfDNA and physiological exercise variables such as peak power output (PPO), running velocity at lactate threshold (LT), and VO₂ max were analyzed. Results cfDNA 90 (10.4-fold, p  < 0.001) and cfDNA 222 (12.4-fold, p  < 0.001) increased significantly after PET. In addition, cfDNA 90 (17.1-fold, p  < 0.001) and cfDNA 222 (20.2-fold, p  < 0.001) increased after HIIT, both remaining significantly elevated 30 min post-HIIT (both p  < 0.001). cfDNA 90 concentrations were higher in afternoon (22.4-fold) compared to morning HIIT sessions (17.2-fold, p  < 0.001). A significant interaction effect was found between group and measurement point for cfDNA 90 ( p  < 0.001) and cfDNA 222 ( p  < 0.001), with higher concentrations in HSM + LIT compared to HSM 30 min post-HIIT. cfDNA 90 showed moderate correlations with PPO ( r  = 0.48, p  < 0.001), LT ( r  = 0.36, p  < 0.001) and VO ₂max ( r  = 0.30, p  = 0.01). cfDNA 222 correlated moderately with VO ₂max ( r  = 0.34, p  = 0.001) and slightly with PPO ( r  = 0.21, p  = 0.05). No chronic changes in cfDNA were observed throughout the study period. Conclusions cfDNA is a reliable marker for detecting acute exercise-induced stress. However, the potential of cfDNA for detecting chronic adaptations in short-term, high-intensity interval training settings, such as a HIIT shock cycle, appears limited thus far. Trial registration clinicaltrials.gov, NCT05067426. Registered 05 October 2021—Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT05067426 . Graphical Abstract Key Points Acute increases in cfDNA concentrations were observed after physiological exercise testing and high-intensity interval training (HIIT). No chronic cfDNA changes were observed during and after a HIIT shock microcycle. Acute increases in cfDNA concentrations correlated with physiological exercise variables e.g. peak power output, running velocity at lactate threshold and VO 2max during acute exercise. Higher increases in cfDNA were found after HIIT compared to physiological exercise testing. Higher increases in cfDNA were found after HIIT in the afternoon compared to morning. Higher increases in cfDNA were observed in males compared to females.

In sepsis, circulating cell-free DNA (cfDNA) originates from host cells (damage-associated molecular patterns, DAMPs) and pathogens (pathogen-associated molecular patterns, PAMPs), contributing to immune activation and offering potential as both a biomarker and a therapeutic target. While DAMPs are thought to predominate in early sepsis, this study aimed to quantify their relative abundance and assess their correlation with inflammatory markers compared to PAMPs. In this prospective observational study, blood samples of 18 ICU patients were collected within 24 hours of sepsis diagnosis. Plasma cfDNA was analyzed via qPCR (targeting human LINE-1) and iSEP-SEQ nanopore sequencing. Human and microbial cfDNA were quantified, and method correlation was assessed using Kendall's tau-b correlation. Associations with inflammatory biomarkers were tested using Spearman correlation analysis and group comparisons between human and non-human reads were analyzed with Pearson correlation analysis. The study received ethical approval from the Landesärztekammer Rheinland-Pfalz (Approval Number: 2020-15535). cfDNA was predominantly of human origin, comprising 99.86% of classified reads, with microbial cfDNA accounting for only 0.077% (p < 0.001). qPCR-based cfDNA concentrations strongly correlated with human read counts from sequencing (τ = 0.712; p < 0.001). Human cfDNA levels were significantly associated with LDH, WBC, and CRP. Microbial cfDNA, although low in abundance, correlated with WBC, CRP, and D-dimer. In early sepsis, human cfDNA is markedly more abundant than microbial cfDNA. However, both exhibit strong correlations with inflammatory and tissue injury markers. These findings support a model of PAMP-triggered and DAMP-driven inflammation and identify human cfDNA as a promising biomarker and potential therapeutic target. https://drks.de/search/de/trial/DRKS00025222/details, identifier DRKS-ID: 00025222.