Cell-free DNA Content Research Articles

Qingjin Huatan decoction attenuates lipopolysaccharide-induced acute lung injury in mice by controlling platelet-associated formation of neutrophil extracellular traps

Abstract Background Acute lung injury (ALI) is a severe and life-threatening lung inflammation with high morbidity and mortality, underscoring the importance to develop effective drugs. Qingjin Huatan decoction (QJHTD), as a classic ancient prescription, has been widely used for treating respiratory diseases. However, the role and mechanism of QJHTD against ALI remain unclear. Objective This study aimed to explore the therapeutic effect of QJHTD on lipopolysaccharide (LPS)-induced ALI in mice and uncover its mechanism. Methods The therapeutic effect of QJHTD on LPS-induced ALI in mice was evaluated by the histopathological changes in the lung tissue, the lung wet/dry weight ratio, and the levels of inflammatory cytokines and thrombin-antithrombin complexes. Transcriptomics was used to predict the mechanism of QJHTD in treating ALI. The expression levels of citrullinated histone 3 in the lung tissue, the content of cell-free DNA in the bronchoalveolar lavage fluid (BALF), and the platelet-associated formation of neutrophil extracellular traps (NETs) in vitro were determined. Results Qingjin Huatan decoction exerted protective effect against LPS-induced ALI by suppressing interstitial edema, maintaining the alveolar-capillary barrier, inhibiting the infiltration of neutrophils and platelets in the lung tissue, and lowering the levels of tumor necrosis factor α, interleukin 1β, interleukin 6, and thrombin-antithrombin complexes in BALF. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis indicated that the formation of NETs was the main regulatory pathway for QJHTD against ALI. Qingjin Huatan decoction could treat ALI by inhibiting the release of NETs via reducing the content of citrullinated histone 3 in lung tissue and cell-free DNA in BALF in vivo, and suppressing the NETs formation induced by LPS-stimulated platelets under flow and static conditions in vitro. The formation of NETs was considered to bridge the interactions between neutrophils and platelets. Conclusions This research demonstrated the effects of QJHTD in treating ALI and provided new insights for clarifying the complex regulation of neutrophils, platelets, and NETs in ALI.

Abstract LB110: Twist pan-cancer synthetic reference materials for cell-free DNA (cfDNA) assay development

Abstract Liquid biopsies hold great promise in reducing the burden of the disease of cancer on patients. Early detection of cancer via the cell free DNA represents the broadest vision and requires highly specificity. Ongoing disease monitoring is another important application of liquid biopsy and requires high sensitivity. Assay development requires analytical validation, and assays must be monitored for ongoing performance. These assays demand reference materials at specific variant allele frequencies for the assessment of specificity and sensitivity. Previously, obtaining reference materials was a laborious process of finding or engineering mutant cell lines. Here, we describe the design, manufacture, and quality control of a synthetic reference standard: the Twist Pan-Cancer Reference Standards, developed using Twist’s proprietary DNA printing platform to include a wide selection of both common and rare cancer targets for analytical validation. The reference material closely mimics the size distribution and content of cell-free DNA, with a primary peak at 167 bp and a secondary peak at 334 bp. The background cfDNA is derived from a single donor and highly characterized through NGS. The reference standard includes over 400 variant sites across 84 genes, including literature-curated, clinically-relevant variant sites. Additional panel-wide variants were included to aid in troubleshooting capture panels. The variant DNA is synthetically printed at 167 bp ± 5 bp and tiles over the site with extensive overlap, providing diversity of DNA termini relative to the position of the site of variation. Variant sites were printed and quantified independently so they can be pooled uniformly. NGS quality controls are implemented at multiple steps to ensure quality. The final products are available in a variant allele frequency (VAF) dilution series. The dilution series is quality controlled using droplet digital PCR (ddPCR) to ensure precision of variant allele frequencies. The Twist Pan Cancer Reference Standards provide a valuable solution for NGS-based assay developers seeking reference materials for a wide array of cancer variants, advancing adoption of liquid biopsy tests toward routine clinical practice. Citation Format: Patrick D. Cherry. Twist pan-cancer synthetic reference materials for cell-free DNA (cfDNA) assay development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB110.

Clinical and pathological features associated with circulating tumor DNA content in real-world patients with metastatic prostate cancer.

BackgroundLiquid biopsy is a powerful tool that can enable treatment decisions for metastatic prostate cancer patients with difficult‐to‐biopsy tumors. However, the detection of genomic alterations via liquid biopsy is limited by the fraction (tumor fraction [TF]) of circulating tumor DNA (ctDNA) within the total cell‐free DNA content. While prior work has preliminarily correlated TF with clinical features of prostate cancer, we sought to validate and provide additional resolution, such that a clinical practitioner might anticipate the probability of successful liquid biopsy profiling leveraging commonly assessed clinical and laboratory features.MethodsA total of 813 liquid biopsy specimens were assessable, with 545 associated with a PSA prostate specific antigen measurement, collected in standard‐of‐care settings across approximately 280 US academic or community‐based cancer clinics from September 2018 to July 2021. Deidentified data were captured into a real‐world clinico‐genomic database (CGDB). Comprehensive genomic profiling (CGP) was performed on extracted cell‐free DNA from liquid biopsy samples.ResultsIn multivariable models, higher PSA level, lower hemoglobin, lower albumin, higher alkaline phosphatase (all p < 0.001), and collection of liquid biopsy blood draw within 60 days of new treatment initiation (p = 0.002) were the most strongly associated features with higher TF. At PSA levels of <5 ng/ml, 43% of patients had a TF of <1% indicating an increased likelihood of unevaluable results. Conversely, at PSA levels of >5 ng/ml, 78% of patients had a TF of at least 1% and 46% had a TF of ≥10%, suggesting improved sensitivity for detection of targetable alterations.ConclusionsUniversal genomic profiling of prostate cancers will require complementary use of liquid biopsy and tumor tissue profiling for suitable patients. The likelihood of adequate ctDNA shedding into plasma is one consideration when deciding whether to pursue CGP via liquid biopsy versus tumor profiling. Our real‐world data suggest that PSA < 5 ng/ml is associated with lower ctDNA yield on liquid biopsy, potentially increasing the incidence of negative results or a need for confirmation with tissue testing.

Identification of a Novel HIF-1α-αMβ2 Integrin-NET Axis in Fibrotic Interstitial Lung Disease.

Neutrophilic inflammation correlates with mortality in fibrotic interstitial lung disease (ILD) particularly in the most severe form, idiopathic pulmonary fibrosis (IPF), although the underlying mechanisms remain unclear. Neutrophil function is modulated by numerous factors, including integrin activation, inflammatory cytokines and hypoxia. Hypoxia has an important role in inflammation and may also contribute to pulmonary disease. We aimed to determine how neutrophil activation occurs in ILD and the relative importance of hypoxia. Using lung biopsies and bronchoalveolar lavage (BAL) fluid from ILD patients we investigated the extent of hypoxia and neutrophil activation in ILD lungs. Then we used ex vivo neutrophils isolated from healthy volunteers and BAL from patients with ILD and non-ILD controls to further investigate aberrant neutrophil activation in hypoxia and ILD. We demonstrate for the first time using intracellular staining, HIF-1α stabilization in neutrophils and endothelial cells in ILD lung biopsies. Hypoxia enhanced both spontaneous (+1.31-fold, p < 0.05) and phorbol 12-myristate 13-acetate (PMA)-induced (+1.65-fold, p < 0.001) neutrophil extracellular trap (NET) release, neutrophil adhesion (+8.8-fold, <0.05), and trans-endothelial migration (+1.9-fold, p < 0.05). Hypoxia also increased neutrophil expression of the αM (+3.1-fold, p < 0.001) and αX (+1.6-fold, p < 0.01) integrin subunits. Interestingly, NET formation was induced by αMβ2 integrin activation and prevented by cation chelation. Finally, we observed NET-like structures in IPF lung sections and in the BAL from ILD patients, and quantification showed increased cell-free DNA content (+5.5-fold, p < 0.01) and MPO-citrullinated histone H3 complexes (+21.9-fold, p < 0.01) in BAL from ILD patients compared to non-ILD controls. In conclusion, HIF-1α upregulation may augment neutrophil recruitment and activation within the lung interstitium through activation of β2 integrins. Our results identify a novel HIF-1α- αMβ2 integrin axis in NET formation for future exploration in therapeutic approaches to fibrotic ILD.

Exosome-encapsulated miR-505 from ox-LDL-treated vascular endothelial cells aggravates atherosclerosis by inducing NET formation.

Neutrophil extracellular traps (NETs) play an important role in the pathological process of atherosclerosis (AS). This study aims to evaluate whether exosomes from oxidized low-density lipoprotein (ox-LDL)-treated vascular endothelial cells (VECs) aggravate AS by inducing NET formation. Exosomes from the peripheral blood of healthy donors and AS patients (namely NC-EXO and AS-EXO, respectively) and exosomes from human umbilical vein endothelial cells (HUVECs) treated without or with ox-LDL (namely normal EXO and ox-LDL-EXO, respectively) were isolated, identified, and co-cultured with neutrophils from peripheral blood of healthy donors. NET formation was evaluated by immunofluorescence staining and determining the content of cell-free DNA and myeloperoxidase-DNA complex. Dual-luciferase reporter assay, chromatin immunoprecipitation assay, quantitative reverse transcription polymerase chain reaction, and western blot analysis were performed to explore the underlying mechanisms. We found that AS-EXO and ox-LDL-EXO induced NET release from neutrophils. Mechanistically, ox-LDL treatment in HUVECs might activate the NF-κB pathway, which transcriptionally activates miR-505, and then the exosome-encapsulated high miR-505 expression targeted and inhibited SIRT3 in neutrophils, thereby inducing reactive oxygen species (ROS) level increase and NET release by neutrophils. Further in vivo experiments showed that ox-LDL-EXO accelerated AS progression in AS mice. In summary, exosome-encapsulated miR-505 from ox-LDL-treated VECs aggravates AS by inducing NET formation.

Увеличение содержания внеклеточной ядерной и митохондриальной ДНК в моче крыс после рентгеновского облучения или введения блеомицина

Purpose: To study the content of cell-free mitochondrial DNA (cf-mtDNA) and cell-free nuclear DNA (cf-nDNA) in urine of rats exposed to ionizing radiation, and after injection of a cytostatic drug bleomycin. Material and methods: Wistar male rats aged 3 months were used in the experiments. Rats were irradiated at a doses of 3, 5, and 8 Gy. Bleomycin was administered intraperitoneally in concentrations of 3, 7, and 10 mg/kg. The DNA content was measured by real-time PCR. Results: The results showed an increase in the level of the number of cf-nDNA and cf-mtDNA fragments in urine of irradiated rats. It was shown that the content of cf-nDNA and cf-mtDNA has a linear dependence on the X-ray dose. Thus, the maximum number of mtDNA and nDNA copies was recorded for 12–24th hours after irradiation. The number of PCR amplification products of cf-mtDNA is 2–3 times higher than those of cf-nDNA. Data analysis of the content of cf-nDNA and cf-mtDNA in rat urine after introduction of bleomycin also showed elevated levels compared with control animals. It was shown that the content of cf-nDNA and cf-mtDNA has a linear dependence on the dose of the chemotherapeutic drug. Conclusion: Thus, it has been shown that it is possible to overcome the transrenal (renal) barrier in animals with cf-mtDNA and cf-nDNA and pass them into the urine after X-ray irradiation, as well as after the administration of bleomycin. The dose dependence of the identified effects was found. The increased content of cell-free DNA in the urine can be considered as a potential biomarker for assessing the level of genotoxic load during radiation damage to the body, as well as when exposed to other genotoxic agents.

Cell-free DNA content in human blastocoel fluid-conditioned media differentiates euploid versus aneuploid embryos

Cell-free DNA content in human blastocoel fluid-conditioned media differentiates euploid versus aneuploid embryos

Expression and significance of neutrophil extracellular traps in ulcerative colitis mouse model

Objective To investigate the expression of neutrophil extracellular traps (NETs) in ulcerative colitis mouse model and its correlation with the degree of intestinal barrier function. Methods Sixty male Balb/c mice were randomly divided into normal group(n=30) and model group(n=30). An ulcerative colitis model was established by dextran sulfate sodium, and intestinal tissue and blood samples were taken on day 1, day 3, day 5 after modeling(10 mice at each time point). By observing the disease activity index and HE pathological analysis, the intestinal tissue damage was reflected.The cell free DNA was quantified using PicoGreen® dye, and the expression and localization of myeloperoxidase and citrullinated histone in the intestine were observed by immunohistochemistry.Western blot was used to detect the expression of Claudin-1. Results Compared with the normal group, the disease activity index in model group increased significantly (P<0.05). The content of cell free DNA increased significantly (P<0.05). Immunohistochemical examination showed that the formation of NETs in the model group increased, and with the disease recovers, its content gradually decreased.Western blot analysis showed that the expression of claudin-1 in the model group decreased (P<0.05), and with the disease recovers, its content gradually increased. Conclusion The formation of NETs increases in the mouse model of ulcerative colitis, and the increased formation of NETs is associated with impairment of intestinal barrier function. Key words: Inflammatory bowel disease; Neutrophil extracellular traps; Tight junction protein

Extracellular DNA in the Dynamics of Uncomplicated Pregnancy.

We analyzed the concentration of extracellular DNA and its fractions in the dynamics of uncomplicated pregnancy Thirty women with singleton pregnancy were examined. The concentration of total, maternal, and fetal cell-free DNA in maternal blood was measured at gestation weeks 11-14, 24-26, and 30-32. The level of total cell-free DNA was evaluated by measuring the concentration of RASSF1A gene using quantitative PCR analysis, the level of cell-free fetal DNA was assessed by determining the hypermethylated part of RASSF1A gene. The concentration of total cell-free DNA and cell-free maternal DNA did not change during the first half of pregnancy, but increased after 24-26 weeks. The level of cell-free fetal DNA increased from the first to the second and third trimester: 14.15 (2.32-36.25), 24.87 (6.29-129.32), and 32.62 (8.97-133.52) GE/ml (p<0.05), respectively. Our results characterize the dynamics of the content of cell-free DNA and its fractions during pregnancy, which should be taken into account when using cell-free DNA for prediction of placenta-associated complications.

Blastocoel cell-free DNA content is related to changes in ploidy status (chromosomal loss/gain) in day-5 embryos

Blastocoel cell-free DNA content is related to changes in ploidy status (chromosomal loss/gain) in day-5 embryos

Circulating cell-free DNA content as blood based biomarker in endometrial cancer.

BackgroundAltered circulating cell-free DNA (cfDNA) levels are related to cancer development and aggressiveness. Up to now, very few studies have been performed for evaluating cfDNA content in endometrial cancer (EC).MethodsFirst, we measured cfDNA release in blood serum of EC cancer patients collected before surgery and before the beginning of any treatment by SYBR Gold assay and correlated it with tumor aggressiveness. We also assessed the relative mitochondrial cell-free DNA (cfmtDNA) content by qRT-PCR. Next, we correlated cfDNA levels with BMI, age, hypertension and inflammation markers.ResultsCfDNA levels are higher in G2 and G3 compared with G1 EC sera. A significant modulation of cfDNA content was detected in sera from patients with BMI>30 compared with those with BMI<30. We observed a further and significant alteration in cfDNA level in hypertensive patients with G2-G3, but not in G1 EC. Analysis of preoperative neutrophil-to-lymphocyte (NLR) and monocyte-to-lymphocyte (MLR) ratios suggests a contribution of the host response in the altered cfDNA levels in EC.ConclusionsOur data indicate that assessment of total and mitochondrial cfDNA levels in blood sera and the relative NLR and MLR in blood obtained from preoperative patients may help clinical management and prognosis in EC.

Experimental study on formation of neutrophil extracellular traps in human peripheral blood induced by paraquat in vitro

To explore whether paraquat (PQ) can induce the formation of neutrophil extracellular traps (NETs) in human peripheral blood. Neutrophils were isolated from healthy human peripheral blood, and the cells were identified by hematoxylin-eosin (HE) strain. The cells were treated with different concentrations of PQ [0 (as control), 200, 400, 600, 800, 1 000 and 1 200 μmol/L], and the cell viability was measured by cell proliferation and CCK-8 cytotoxicity detection kit, and the median lethal concentration of PQ was selected. The cells were treated with the median lethal concentration of PQ (PQ poisoning group), and the untreated cells were served as the control. Immunofluorescence staining was adopted to evaluate NETs formation. PicoGreen dye was used to determine the quantitative content of circulating free DNA. Western Blot was used to determine the expressions of citrullinated histone 3 (H3Cit) and myeloperoxidase (MPO) in the supernatant. The purity of neutrophils was about 95% by HE staining. The cells were treated with different concentrations of PQ, and the result showed that the viability of cells was (58±2)% with 800 μmol/L PQ for treatment. The immunofluorescence showed that there were few expressions of H3Cit and MPO in neutrophils in the control group, and there was no NETs formation, which was composed of DNA, H3Cit and MPO. Compared with the control group, a large amount of NETs was generated from neutrophils stimulated by 800 μmol/L of PQ. Meanwhile, quantitative result showed that the content of cell free DNA in the supernatant was significantly increased in PQ poisoning group as compared with that of control group (μg/L: 2 235±462 vs. 561±87, P < 0.01). The protein expressions of H3Cit and MPO in the supernatant were also significantly increased as compared with those of control group [H3Cit protein expression (gray value): 0.23±0.03 vs. 0.11±0.01, MPO protein expression (gray value): 0.47±0.05 vs. 0.21±0.04, both P < 0.05]. 800 μmol/L of PQ can induce the formation of NETs in human peripheral blood.