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Germ cell tumor (GCT) patients with unfavourable response to first-line therapy still lack reliable diagnostic and effective treatment Detailed correlation of total extracellular DNA (ecDNA), other DNA species and endogenous DNase levels in GCT patients' plasma and translational utility remains under- investigated. We determined DNase plasma levels, ecDNA of different subcellular origin and neutrophil extracellular trap (NETs)-associated markers. Next, we determined the associations of these parameters with a level of the DNA damage, immune inflammatory index, specific immune cell subpopulations in a cohort of the 117 GCT patients and 19 matched healthy donors (HDs). Moreover, we investigated how exogenous DNase affects antitumor effect of cisplatin in GCT model of cisplatin-resistant embryonal carcinoma NTERA-2 CisR. Our data demonstrate that high level of ecDNA and low level of DNase in GCT patients' plasma is associated with significantly worse progression-free survival and overall survival. The level of the plasma ecDNA was five times higher in the GCT patients compared to the HDs. The patients with higher total ecDNA and ncDNA, but not mtDNA, had inferior PFS and OS compared to the patients with lower ecDNA (all p < 0.05). There was an inverse correlation between plasma DNase and ecDNA levels, and between plasma DNase level and clinical outcome. Importantly, combined treatment with cisplatin and human recombinant DNase I delayed growth of the NTERA-2 CisR xenografts and prolonged animal survival. Importantly, Pulmozyme significantly reduced intratumoral microvascular density in our preclinical model. Our data confirm the association between low plasma DNase activity and worse overall survival for the first time in GCT patients. This study further validated the prognostic value of total ecDNA in GCT patients. More importantly, our preclinical data substantiated beneficial effect of Pulmozyme combination with cisplatin treatment to improve the therapeutic outcome in refractory disease.

[This corrects the article DOI: 10.1371/journal.pone.0336190.].

This study aimed to evaluate the amniotic fluid protein profiles and the intensity of intraamniotic inflammatory response to Ureaplasma spp. and other bacteria, using the multiplex xMAP technology. A retrospective cohort study was undertaken in the Department of Obstetrics and Gynecology, University Hospital Hradec Kralove, Czech Republic. A total of 145 pregnant women with preterm prelabor rupture of membranes between gestational age 24+0 and 36+6 weeks were included in the study. Amniocenteses were performed. The presence of Ureaplasma spp. and other bacteria was evaluated using 16S rRNA gene sequencing. The levels of specific proteins were determined using multiplex xMAP technology. The presence of Ureaplasma spp. and other bacteria in the amniotic fluid was associated with increased levels of interleukin (IL)-6, IL-8, IL-10, brain-derived neurotropic factor, granulocyte macrophage colony stimulating factor, monocyte chemotactic protein-1, macrophage inflammatory protein-1, and matrix metalloproteinasis-9. Ureaplasma spp. were also associated with increased levels of neurotropin-3 and triggering receptor expressed on myeloid cells-1. The presence of Ureaplasma spp. in the amniotic fluid is associated with a slightly different protein profile of inflammatory response, but the intensity of inflammatory response to Ureaplasma spp. is comparable with the inflammatory response to other bacteria.

Cisplatin-induced peripheral neuropathy (CIPN) is a frequent and often persistent complication in survivors of testicular germ cell tumors (GCT) treated with curative therapy. Although several mechanisms have been proposed, the biological drivers of long-term neurotoxicity remain incompletely understood. Disruption of intestinal barrier integrity during chemotherapy or radiotherapy may promote gut microbial translocation (GMT), leading to systemic immune activation and chronic inflammation that could contribute to neuropathy development. This study investigated the relationship between circulating biomarkers of GMT and symptoms of CIPN in long-term GCT survivors. A total of 170 GCT survivors (median age 41 years) from the National Cancer Institute of Slovakia were included, with a median follow-up of 10 years after treatment. Participants completed the EORTC QLQ-CIPN20 questionnaire assessing sensory, motor, and autonomic neuropathy. Peripheral blood samples were analyzed for plasma biomarkers associated with gut microbial translocation and innate immune activation, including soluble CD14 (sCD14), high-mobility group box-1 (HMGB1), lipopolysaccharide (LPS), and D-lactate. Associations between biomarker concentrations and CIPN scores were evaluated across treatment groups: orchiectomy only (active surveillance, n=28), cisplatin-based chemotherapy (n=119), radiotherapy (n=14), and combined chemoradiotherapy (n=9). Patients with higher plasma sCD14 levels had significantly higher overall CIPN scores (7.6% increase, p=0.019) and worse sensory function (9.5% increase, p=0.019) compared with those with lower levels. Patients treated with chemotherapy exhibited significantly higher plasma sCD14 levels than those under active surveillance (6613 vs. 3768 μg/L, p = 0.009). Among chemotherapy-treated patients, elevated sCD14 was associated with a higher risk of motor neuropathy (RR = 3.5, 95% CI 1.21-10.14, p=0.020). In survivors receiving combined chemotherapy and radiotherapy, increased sCD14 levels were associated with a higher risk of autonomic neuropathy (RR = 2.85, 95% CI 1.11-7.30, p=0.029). Elevated HMGB1 was also associated with an increased probability of autonomic dysfunction (RR = 2.15, 95% CI 1.07-4.33, p=0.015). No significant associations were observed between cumulative cisplatin dose and GMT biomarkers. Elevated biomarkers of gut microbial translocation, particularly sCD14, are associated with increased severity of CIPN in long-term GCT survivors. These findings support the hypothesis that treatment-related intestinal barrier disruption and subsequent immune activation may contribute to persistent neurotoxicity in cancer survivorship.

Extracellular DNA, also called cell-free DNA, released from dying cells or activated immune cells can be recognized by the immune system as a danger signal causing or enhancing inflammation. The cleavage of extracellular DNA is crucial for limiting the inflammatory response and maintaining homeostasis. Deoxyribonucleases (DNases) as enzymes that degrade DNA are hypothesized to play a key role in this process as a determinant of the variable concentration of extracellular DNA. DNases are divided into two families—DNase I and DNase II, according to their biochemical and biological properties as well as the tissue-specific production. Studies have shown that low DNase activity is both, a biomarker and a pathogenic factor in systemic lupus erythematosus. Interventional experiments proved that administration of exogenous DNase has beneficial effects in inflammatory diseases. Recombinant human DNase reduces mucus viscosity in lungs and is used for the treatment of patients with cystic fibrosis. This review summarizes the currently available published data about DNases, their activity as a potential biomarker and methods used for their assessment. An overview of the experiments with systemic administration of DNase is also included. Whether low-plasma DNase activity is involved in the etiopathogenesis of diseases remains unknown and needs to be elucidated.

Background Patients with ovarian cancer represent a heterogeneous population with a variable prognosis and response to chemotherapy. Plasma DNA has been shown to have a prognostic value in different types of cancer including ovarian carcinoma. Whether total circulating DNA, which can be assessed much easier without knowing the tumor-specific mutations, has similar informative value is currently unknown. The aim of this study was to evaluate the prognostic value of extracellular DNA in advanced ovarian cancer. Methods This prospective study included 67 patients (pts) with ovarian cancer treated with 1st line paclitaxel and carboplatin (25 pts) and paclitaxel, carboplatin and bevacizumab (42 pts). Thirty-five patients had optimal surgical debulking before chemotherapy. Extracellular DNA was quantified using real time PCR before administration of chemotherapy (67 pts) and after 6 cycles of chemotherapy (44 pts). Results Total extracellular DNA (ecDNA), as well as extracellular DNA of nuclear (nDNA) and mitochondrial origin (mtDNA) significantly ( p  < 0.05) decreased after 6 cycles of chemotherapy (by 54%, 63% and 52%, respectively. Patients with stage I disease had significantly lower mtDNA compared to patients with stage II-IV (8604 vs. 16, 984 ge/mL, p  = 0.03). Patients with lower baseline nDNA had superior progression-free (HR = 0.35 (0.14–0.86)) and overall survival (HR = 0.18 (0.04–0.77). The prognostic value of nDNA was confirmed independent of tumor stage and confirmed in multivariate analysis. Conclusions Our data suggest that ecDNA of both, nuclear and mitochondrial origin could be added to prognostic markers in ovarian cancer. Analysis of ecDNA does not require the knowledge of tumor-specific mutations in contrast to the quantification of tumor-derived ecDNA. Study of the dynamics and cell type-specific source of the ecDNA could shed light on its biology in cancer and might help to direct the treatment of ovarian cancer.

Resveratrol is a natural polyphenol studied for its possible protective properties in inflammatory bowel diseases. Moreover, it has been shown to interact with estrogen receptors. In the present study, we aimed to investigate possible diverse effects of resveratrol on female and male mice in DSS-induced colitis. Thirty-seven C57BL/6 mice (21 female and 16 male) were divided into three groups for each sex. The first group received pure water (CTRL). The other two groups received 1.5% dextran sulfate sodium (DSS) to induce colitis from which one group was treated with resveratrol (DSS + RSV). Intake of 1.5% DSS caused weight loss in all DSS groups compared to control mice. Weight loss, stool consistency, and discomfort did not show any protective effect of resveratrol in males and showed even adverse effects in females. In females, the activity of myeloperoxidase was lower compared to that in males. However, colon length and spleen weight showed no sex differences, which can indicate the induction of only mild colitis in mice. Resveratrol did not have any effect on TNF-alpha levels. Taken together, these results for the first time propose possible diverse effects of resveratrol in DSS-induced colitis model depending on the sex of the animal. However, this conclusion must be confirmed by further analyses.

Cell-free DNA (cfDNA) is present in various body fluids and originates mostly from blood cells. In specific conditions, circulating cfDNA might be derived from tumours, donor organs after transplantation or from the foetus during pregnancy. The analysis of cfDNA is mainly used for genetic analyses of the source tissue —tumour, foetus or for the early detection of graft rejection. It might serve also as a nonspecific biomarker of tissue damage in critical care medicine. In kidney diseases, cfDNA increases during haemodialysis and indicates cell damage. In patients with renal cell carcinoma, cfDNA in plasma and its integrity is studied for monitoring of tumour growth, the effects of chemotherapy and for prognosis. Urinary cfDNA is highly fragmented, but the technical hurdles can now be overcome and urinary cfDNA is being evaluated as a potential biomarker of renal injury and urinary tract tumours. Beyond its diagnostic application, cfDNA might also be involved in the pathogenesis of diseases affecting the kidneys as shown for systemic lupus, sepsis and some pregnancy-related pathologies. Recent data suggest that increased cfDNA is associated with acute kidney injury. In this review, we discuss the biological characteristics, sources of cfDNA, its potential use as a biomarker as well as its role in the pathogenesis of renal and urinary diseases.

The aims of this study were to test the utility of benchtop NGS platforms for NIPT for trisomy 21 using previously published z score calculation methods and to optimize the sample preparation and data analysis with use of in silico and physical size selection methods. Samples from 130 pregnant women were analyzed by whole genome sequencing on benchtop NGS systems Ion Torrent PGM and MiSeq. The targeted yield of 3 million raw reads on each platform was used for z score calculation. The impact of in silico and physical size selection on analytical performance of the test was studied. Using a z score value of 3 as the cut-off, 98.11%-100% (104-106/106) specificity and 100% (24/24) sensitivity and 99.06%-100% (105-106/106) specificity and 100% (24/24) sensitivity were observed for Ion Torrent PGM and MiSeq, respectively. After in silico based size selection both platforms reached 100% specificity and sensitivity. Following the physical size selection z scores of tested trisomic samples increased significantly--p = 0.0141 and p = 0.025 for Ion Torrent PGM and MiSeq, respectively. Noninvasive prenatal testing for chromosome 21 trisomy with the utilization of benchtop NGS systems led to results equivalent to previously published studies performed on high-to-ultrahigh throughput NGS systems. The in silico size selection led to higher specificity of the test. Physical size selection performed on isolated DNA led to significant increase in z scores. The observed results could represent a basis for increasing of cost effectiveness of the test and thus help with its penetration worldwide.

Rheumatoid arthritis (RA) as a chronic autoimmune inflammatory disease increases extracellular DNA (ecDNA). Our previous study has shown that anti-inflammatory treatment reduces ecDNA, but it is unclear whether there is an association with treatment response. The aim of this study was to analyze the changes of ecDNA induced by biological disease-modifying antirheumatic drugs (bDMARDs) in RA patients with an emphasis on the subcellular origin of ecDNA. Plasma samples from 40 RA patients were collected in three different time-points: before treatment with bDMARDs as well as 3 and 12 months following treatment initiation. Total, nuclear and mitochondrial ecDNA was quantified using fluorometry and real-time PCR. Disease activity score (DAS28) and C-reactive protein (CRP) were used to monitor the clinical status and the response to treatment. Treatment with bDMARDs elicited an overall improvement of the clinical status: DAS28 and CRP showed a significant decrease by 54% and 43%, respectively, after 3 months of treatment. A significant decrease of total ecDNA by 60% and nuclear ecDNA by 58% was detected only in good responders after 3 months of bDMARDs treatment. No significant changes of plasma ecDNA concentration were observed in moderate and non-responders. Deoxyribonuclease activity was not affected by the treatment. None of the analyzed biomarkers differed between the groups at baseline. Plasma ecDNA especially of nuclear origin could potentially be useful to monitor the treatment response in RA. Further studies should shed light on disease-treatment interplay implicated in ecDNA origin potentially linked to neutrophil extracellular traps.