Cationic mesoporous silica nanoparticles alleviate osteoarthritis by targeting multiple inflammatory mediators.

Osteoarthritis (OA) is a common and complex inflammatory disorder that is frequently compounded by cartilage degradation, synovial inflammation, and osteophyte formation. Damaged chondrocytes release multiple danger mediators that exacerbate synovial inflammation and accelerate the progression to OA. Conventional treatments targeting only a single mediator of OA have failed to achieve a strong therapeutic effect. Addressing the crucial role of multiple danger mediators in OA progression, we prepared polyethylenimine (PEI)-functionalized diselenide-bridged mesoporous silica nanoparticles (MSN-PEI) with cell-free DNA (cfDNA)-binding and anti-oxidative properties. In models of surgery-induced and collagenase-induced arthritis, we showed that these cationic nanoparticles attenuated cartilage degradation and provided strong chondroprotection against joint damage. Mechanistically, multiple target blockades alleviated oxidative stress and dampened cfDNA-induced inflammation by suppressing the M1 polarization of macrophages. This study suggests a beneficial direction for targeting multiple danger mediators in the treatment of intractable arthritis.

Cell-free fetal DNA impairs trophoblast migration in a TLR9-dependent manner and can be reversed by hydroxychloroquine.

Growing evidence suggests a relationship between elevated circulating placental-derived cell-free fetal DNA (cffDNA) and preeclampsia. Hypomethylation of CpG motifs, a hallmark of cffDNA, allows it to activate Toll-like receptor 9 (TLR9). Using an in vitro human first trimester extravillous trophoblast cell model, we sought to determine if trophoblast-derived cffDNA and ODN 2216, a synthetic unmethylated CpG oligodeoxynucleotide, directly impacted spontaneous trophoblast migration. The role of the DNA sensors TLR9, AIM2, and cGAS was assessed using the inhibitor A151. To test whether any effects could be reversed by therapeutic agents, trophoblasts were treated with or without cffDNA or ODN 2216 with or without aspirin (ASA; a known cGAS inhibitor), aspirin-triggered lipoxin (ATL), or hydroxychloroquine (HCQ; a known TLR9 inhibitor). Trophoblast-derived cffDNA and ODN 2216 reduced trophoblast migration without affecting cell viability. Reduced trophoblast migration in response to cffDNA or ODN 2216 was reversed by A151. cffDNA inhibition of trophoblast migration was reversed by HCQ, while ASA or ATL had no effect. In contrast ODN 2216 inhibition of trophoblast migration was reversed by ASA, ATL and HCQ. Our findings suggest that cffDNA can exert a local effect on placental function by impairing trophoblast migration through activation of innate immune DNA sensors. HCQ, a known TLR9 inhibitor, reversed the effects of cffDNA on trophoblast migration. Greater insights into the molecular underpinnings of how cffDNA impacts placentation can aid in our understanding of the pathogenesis of preeclampsia, and in the development of novel therapeutic approaches for preeclampsia therapy.

Nucleic Acid-Scavenging Hydrogels Accelerate Diabetic Wound Healing.

Chronic inflammation is a typical feature and a major impediment in refractory diabetic foot ulcer (DFU). High levels of extracellular cell-free nucleic acid (cfDNA) have recently been known to play a critical role in the cause of inflammation. Herein, we fabricated polyacrylamide-based cationic hydrogels and topically applied them to the ulcer of a diabetic rat model. The cfDNA level in the wound area was significantly reduced after hydrogel adsorption, and the level of inflammation was eliminated. In turn, the wound closure was significantly promoted without introducing systemic toxicity. Cationic hydrogels represent an effective material to combat uncontrolled inflammation in DFU.

Insight into diagnosis of pleural tuberculosis with special focus on nucleic acid amplification tests.

INTRODUCTION: Pleural tuberculosis (TB) is the archetype of extrapulmonary TB (EPTB), which mainly affects the pleural space and leads to exudative pleural effusion. Diagnosis of pleural TB is a difficult task predominantly due to atypical clinical presentations and sparse bacillary load in clinical specimens. AREA COVERED: We reviewed the current literature on the globally existing conventional/latest modalities for diagnosing pleural TB. Bacteriological examination (smear/culture), tuberculin skin testing/interferon-γ release assays, biochemical testing, imaging and histopathological/cytological examination are the main modalities. Moreover, nucleic acid amplification tests (NAATs), i.e. loop-mediated isothermal amplification, PCR/multiplex-PCR, nested-PCR, real-time PCR and GeneXpert® MTB/RIF are being utilized. Currently, GeneXpert Ultra, Truenat MTBTM, detection of circulating Mycobacterium tuberculosis (Mtb) cell-free DNA by NAATs, aptamer-linked immobilized sorbent assay and immuno-PCR (I-PCR) have also been exploited. EXPERT OPINION: Routine tests are not adequate for effective pleural TB diagnosis. The latest molecular/immunological tests as discussed above, and the other tools, i.e. real-time I-PCR/nanoparticle-based I-PCR and identification of Mtb biomarkers within urinary/serum extracellular vesicles being utilized for pulmonary TB and other EPTB types may also be explored to diagnose pleural TB. Reliable diagnosis and early therapy would reduce the serious complications associated with pleural TB, i.e. TB empyema, pleural fibrosis, etc.

Circulating cell free DNA and citrullinated histone H3 as useful biomarkers of NETosis in endometrial cancer.

BACKGROUND: Cancer mortality is mainly caused by organ failure and thrombotic events. It has been demonstrated that NETosis, a chromatin release mechanism implemented by neutrophils, may contribute to these lethal systemic effects. Our aim was to investigate NETosis biomarkers in endometrial cancer (EC). METHODS: The experiments were conducted on 21 healthy subjects (HS) with no gynecological conditions, and on 63 EC patients. To assess the presence of NETosis features, IHC and IF was performed using antibodies against citrullinated histone H3 (citH3), neutrophil elastase (NE) and histone 2B. Serum levels of cell free DNA (cfDNA), cell free mitochondrial DNA (cfmtDNA) and citH3 were measured by qPCR using one microliter of deactivated serum, and by ELISA assay respectively. Fragmentation pattern of serum cfDNA was analyzed using the Agilent 2100 Bioanalyzer and High Sensitivity DNA Chips. Receiver operating characteristic (ROC) analysis was used to identify a cut off for cfDNA and cfmtDNA values able to discriminate between ECs and HSs. Correlation analysis and multiple correspondence analysis (MCA) between cfDNA, mtcfDNA, citH3 and blood parameters were used to identify the potential association among serum parameters in EC grades. RESULTS: We demonstrated the presence of NETosis features in tissues from all EC grades. Serum cfDNA and cfmtDNA levels discriminate ECs from HSs and a direct correlation between citH3 and cfDNA content and an inverse correlation between cfmtDNA and citH3 in EC sera was observed, not detectable in HSs. MCA indicates cfDNA, cfmtDNA and citH3 as features associated to G1 and G2 grades. A correlation between increased levels of cfDNA, citH3 and inflammation features was found. Finally, serum nucleosomal cfDNA fragmentation pattern varies in EC sera and correlates with increased levels of cfDNA, citH3, lymphocytes and fibrinogen. CONCLUSION: Our data highlight the occurrence of NETosis in EC and indicate serum cfDNA and citH3 as noninvasive biomarkers of tumor-induced systemic effects in endometrial cancer.

Post-treatment cell-free DNA as a predictive biomarker in molecular-targeted therapy of hepatocellular carcinoma.

BACKGROUND: Liquid biopsies, particularly those involving circulating tumor DNA (ctDNA), are rapidly emerging as a non-invasive alternative to tumor biopsies. However, clinical applications of ctDNA analysis in hepatocellular carcinoma (HCC) have not been fully elucidated. METHODS: We measured the amount of plasma-derived cell-free DNA (cfDNA) in HCC patients before (n = 100) and a few days after treatment (n = 87), including radiofrequency ablation, transarterial chemoembolization, and molecular-targeted agents (MTAs), and prospectively analyzed their associations with clinical parameters and prognosis. TERT promoter mutations in cfDNA were analyzed using droplet digital PCR. Furthermore, we performed a comprehensive mutational analysis of post-treatment cfDNA via targeted ultra-deep sequencing (22,000× coverage) in a panel of 275 cancer-related genes in selected patients. RESULTS: Plasma cfDNA levels increased significantly according to HCC clinical stage, and a high cfDNA level was independently associated with a poor prognosis. TERT promoter mutations were detected in 45% of all cases but were not associated with any clinical characteristics. cfDNA levels increased significantly a few days after treatment, and a greater increase in post-treatment cfDNA levels was associated with a greater therapeutic response to MTAs. The detection rate of TERT mutations increased to 57% using post-treatment cfDNA, suggesting that the ctDNA was enriched. Targeted ultra-deep sequencing using post-treatment cfDNA after administering lenvatinib successfully detected various gene mutations and obtained promising results in lenvatinib-responsive cases. CONCLUSIONS: Post-treatment cfDNA analysis may facilitate the construction of biomarkers for predicting MTA treatment effects.

Utility of Cerebrospinal Fluid Cell-Free DNA in Patients with EGFR-Mutant Non-Small-Cell Lung Cancer with Leptomeningeal Metastasis.

BACKGROUND: Leptomeningeal metastasis (LM) is a fatal complication of advanced non-small-cell lung cancer (NSCLC). OBJECTIVE: The aim of this study was to evaluate the utility of cerebrospinal fluid (CSF) as a medium for epidermal growth factor receptor (EGFR) mutation testing in clinical practice. PATIENTS AND METHODS: We prospectively enrolled patients with EGFR-mutant NSCLC who underwent CSF sampling for suspected LM. The supernatant of CSF after routine cytology examination was collected. The diagnosis of LM was established according to EANO-ESMO criteria. CSF and plasma cell-free DNA (cfDNA) were retrieved for EGFR mutation testing. RESULTS: Fifty-one patients with a median age of 62.7 years were enrolled. The median duration from initial diagnosis to CSF sampling was 23.0 months and most patients (94.1%) had received at least one EGFR-tyrosine kinase inhibitor. Adenocarcinoma cells were found in 37 CSF samples (72.5%), and 48 (94.1%) patients had confirmed or probable LM. Thirty-five of these 48 patients (72.9%) had valid EGFR mutation-testing results using CSF cfDNA and tended to have higher white blood cell counts and positive cytology in their CSF compared to those with invalid mutation testing results. The overall detection rate of EGFR mutation in CSF cfDNA was 68.8%, and the T790M detection rate was 14.6%. In 37 patients with paired CSF and plasma samples, the concordance rate of the EGFR mutation results was 29.7%. CONCLUSIONS: For patients with EGFR-mutant NSCLC with LM, CSF supernatant is a valuable source for EGFR mutation testing and may provide important information.

A novel pathway for the induction of DNA damage in human spermatozoa involving extracellular cell-free DNA.

DNA damage is a common feature of human spermatozoa associated with an impaired capacity to fertilize the oocyte and an increased mutational load in the offspring. However, the etiology of this damage remains poorly defined. In this study we demonstrate that a major pathway for the induction of DNA damage in mammalian spermatozoa is triggered by exposure to exogenous cell free DNA (cfDNA). Exposure of human and mouse spermatozoa to cfDNA (calf thymus, mouse liver and salmon testes) in vitro induced a dose-dependent increase in sperm DNA damage that could be effectively suppressed by the concomitant presence of DNase. The induction of such damage was not accompanied by any concomitant change in sperm motility or vitality and was not directly associated with the induction of oxidative stress. In vivo the injection of exogenous DNA again precipitated an increase in sperm DNA fragmentation that could be reversed by the prior administration of DNase. Similarly, the induction of a transient unilateral testicular ischemia induced an increase in DNA fragmentation that was evident within 24 h and sustained for at least 14 days via mechanisms that could be completely suppressed by the prior administration of DNase. We conclude that exogenous cfDNA activates a defensive response in human spermatozoa associated with the nuclease-mediated induction of DNA fragmentation, possibly involving the participation of TLR9 and CD4. These novel insights have significant implications for our understanding of DNA fragmentation in the male germ line and open up new pathways for the remediation of this condition.

Identification of Specific Joint-Inflammatogenic Cell-Free DNA Molecules From Synovial Fluids of Patients With Rheumatoid Arthritis.

Elevated cell-free DNA (cfDNA) levels in the plasma and synovial fluid of rheumatoid arthritis (RA) patients are proposed to be pathologically relevant. However, direct evidence to support this perception is lacking, and molecular feature of the cfDNA molecules with assumed pathological function is not well characterized. Here, we confirm remarkably increased levels of total synovial fluid and plasma cfDNAs in a large cohort of patients with rheumatoid arthritis compared to the counterparts in osteoarthritis, and demonstrate the potent inflammatogenic effects of RA synovial fluid cfDNA on both human monocyte cell line and primary cells related to RA. Massively parallel sequencing identifies distinct molecular pattern of cfDNA in RA, as characterized by enriching CpG-motif containing sequences. Importantly, these identified CpG-motif-rich sequences are hypomethylated in RA patients and induce severe inflammatory responses both in vitro and in vivo. Our data demonstrate the pathological role of global and specific cfDNA molecules in RA, thereby identifying novel therapeutic target candidate and potential biomarker for RA.

Immunoregulatory properties of cell-free DNA in plasma of celiac disease patients - A pilot study.

The elevated plasma cell-free DNA (cfDNA) concentrations were repeatedly reported in association with the process of inflammation. The qualitative and quantitative characteristics of plasma cfDNA in active (newly diagnosed) celiac disease patients (CD) have not yet been studied despite the fact that cfDNA of healthy individuals is able to regulate immune response. We determined the total cfDNA concentration and relative content of telomeric sequences in plasma cfDNA in CD (n = 10) and healthy age- and sex-matched controls (HC, n = 10) by quantitative PCR. To obtain the evidence that the observed biological effects are caused solely by cfDNA molecules, we applied the treatment of paired plasma samples with DNase. Using paired samples of plasma (non-treated/native and treated by DNase), we analyzed the contribution of cfDNA to the activation of TLR9 and TNF-α mRNA expression in THP1 monocytic cell line. There were no significant differences in the quantities of plasma cfDNA and relative contents of telomeric sequences in their pools. When we compared the levels of TNF-α mRNA expression in THP1 cells achieved after stimulation with native CD and HC plasma samples, we found significantly (p = .031) higher expression after stimulation with CD samples. We documented also the ability of cfDNA contained in CD plasma samples to stimulate the production of TLR9 mRNA. The TLR9 mRNA expression levels were significantly (p = .014) lowered after cfDNA removal from CD plasma samples. The design of our experiments allowed us to study the effects of cfDNA without its isolation from plasma. cfDNA contained in CD plasma samples differs significantly in its immunoregulatory capacity from cfDNA in HC plasma. The differences are caused neither by different concentrations of cfDNA in plasma samples nor by different relative abundance of telomeric sequences. Further studies are needed to elucidate the role of plasma cfDNA in celiac disease pathogenesis.

Clinical Utility of Cerebrospinal Fluid Cell-Free DNA as Liquid Biopsy for Leptomeningeal Metastases in ALK-Rearranged NSCLC.

INTRODUCTION: Leptomeningeal metastases (LMs) indicated a poor prognosis in NSCLC. LMs were more frequent in driver gene-mutated patients, and cerebrospinal fluid (CSF) cell-free DNA has shown unique genetic profiles of LM in EGFR-mutated LM. However, studies in patients with ALK receptor tyrosine kinase gene (ALK)-rearranged NSCLC with LMs are scarce. METHODS: Patients with lung cancer with ALK rearrangement were screened from September 2011 to February 2018 at our institute. CSF and paired plasma were tested by next-generation sequencing. RESULTS: LMs were diagnosed in 30 (10.3%) of 291 patients with ALK-rearranged lung cancer. A total of 11 paired CSF and plasma samples tested by next-generation sequencing were analyzed. Driver genes were detected in 81.8% of the CSF samples (9 of 11) and 45.5% of the plasma samples (5 of 11) (p = 0.183). The maximum allelic fractions were all higher in CSF than in plasma (p = 0.009). ALK and tumor protein p53 gene (TP53) were the two most frequently mutated genes in CSF. Gatekeeper gene ALK G1202R and C1156F mutations were identified in CSF after resistance to alectinib. Multiple copy number variants were mainly found in CSF, including in EGFR, cyclin D1 gene (CCND1), fibroblast growth factor 3 gene (FGF3), and fibroblast growth factor 4 gene (FGF4). Also found were v-myc avian myelocytomatosis viral oncogene homolog gene (MYC) copy number gains and TP53 and cyclin dependent kinase inhibitor 2A gene (CDKN2A) copy number deletions. Brigatinib seemed to be effective in controlling LM. One case showed that CSF could be used to monitor disease development of LM and longitudinally monitor tumor response. CONCLUSION: Liquid biopsy of CSF is more sensitive than liquid biopsy of plasma to detect targetable alterations, characterizing resistance mechanisms on progression and monitoring tumor response in patients with ALK-rearranged NSCLC with LM. Thus, CSF might be promising as a medium of liquid biopsy in LM.

The myocardial infarct-exacerbating effect of cell-free DNA is mediated by the high-mobility group box 1-receptor for advanced glycation end products-Toll-like receptor 9 pathway.

INTRODUCTION: Damage-associated molecular patterns, such as high-mobility group box 1 (HMGB1) and cell-free DNA (cfDNA), play critical roles in mediating ischemia-reperfusion injury (IRI). HMGB1 activates RAGE to exacerbate IRI, but the mechanism underlying cfDNA-induced myocardial IRI remains unknown. We hypothesized that the infarct-exacerbating effect of cfDNA is mediated by HMGB1 and receptor for advanced glycation end products (RAGE). METHODS: C57BL/6 wild type mice, RAGE knockout (KO), and Toll-like receptor 9 KO mice underwent 20- or 40-minute occlusions of the left coronary artery followed by up to 60 minutes of reperfusion. Cardiac coronary perfusate was acquired from ischemic hearts without reperfusion. Exogenous mitochondrial DNA was acquired from livers of normal C57BL/6 mice. Myocardial infarct size (IS) was reported as percent risk region, as measured by 2,3,5-triphenyltetrazolium chloride and Phthalo blue (Heucotech, Fairless Hill, Pa) staining. cfDNA levels were measured by Sytox Green assay (Thermo Fisher Scientific, Waltham, Mass) and/or spectrophotometer. RESULTS: Free HMGB1 and cfDNA levels were increased in the ischemic myocardium during prolonged ischemia and subsequently in the plasma during reperfusion. In C57BL/6 mice undergoing 40'/60' IRI, deoxyribonuclease I, or anti-HMGB1 monoclonal antibody reduced IS by approximately half to 29.0% ± 5.2% and 24.3% ± 3.5% (P < .05 vs control 54.3% ± 3.4%). However, combined treatment with deoxyribonuclease I + anti-HMGB1 monoclonal antibody did not further attenuate IS (29.3% ± 4.9%). In C57BL/6 mice undergoing 20'/60' IRI, injection of 40'/5' plasma upon reperfusion increased IS by more than 3-fold (to 19.9 ± 4.3; P < .05). This IS exacerbation was abolished by pretreating the plasma with deoxyribonuclease I or by depleting the HMGB1 by immunoprecipitation, or by splenectomy. The infarct-exacerbating effect also disappeared in RAGE KO mice and Toll-like receptor 9 KO mice. Injection of 40'/0' coronary perfusate upon reperfusion similarly increased IS. The levels of HMGB1 and cfDNA were significantly elevated in the 40'/0' coronary perfusate and 40'/reperfusion (min) plasma but not in those with 10' ischemia. In C57BL/6 mice without IRI, 40'/5' plasma significantly increased the interleukin-1ß protein and messenger RNA expression in the spleen by 30 minutes after injection. Intravenous bolus injection of recombinant HMGB1 (0.1 µg/g) or mitochondrial DNA (0.5 µg/g) 5 minutes before reperfusion did not exacerbate IS (P = not significant vs control). However, combined administration of recombinant HMGB1 + mitochondrial DNA significantly increased IS (P < .05 vs individual treated groups) and this infarct-exacerbating effect disappeared in RAGE KO mice and splenectomized C57BL/6 mice. The accumulation of cfDNA in the spleen after combined recombinant HMGB1 + mitochondrial DNA treatment was significantly more elevated in C57BL/6 mice than in RAGE KO mice. CONCLUSIONS: Both HMGB1 and cfDNA are released from the heart upon reperfusion after prolonged ischemia and both contribute importantly and interdependently to post-IRI by a common RAGE-Toll-like receptor 9-dependent mechanism. Depleting either of these 2 damage-associated molecular patterns suffices to significantly reduce IS by approximately 50%.

STRECK CELL PRESERVATIVE STABILIZES KOALA ( PHASCOLARCTOS CINEREUS) WHOLE BLOOD FOR COMPLETE BLOOD COUNTS.

Wildlife health assessments at remote sites may lead to delayed testing of whole blood for complete blood counts (CBC) resulting in artifacts affecting clinical interpretation. Streck Cell Preservative (SCP) is a proprietary liquid stabilization reagent designed to preserve human leukocytes and may be applicable to wildlife health assessments when immediate processing of blood is not possible. The purpose of this study was to determine if SCP adequately preserved EDTA-anticoagulated whole blood from koalas ( Phascolarctos cinereus) for up to 14 days. Blood from 12 captive adult koalas was collected and combined with SCP in a 1 : 1 ratio and refrigerated. Paired samples of SCP treated and untreated blood had CBCs performed at five time-points over 14 days. Streck Cell Preservative extended koala EDTA-anticoagulated whole blood viability to 14 days by decreasing cellular lysis. Species- and method-specific reference intervals for SCP should be generated to avoid clinical misinterpretation, especially when evaluating anemia.

Cell-free DNA induced apoptosis of granulosa cells by oxidative stress.

BACKGROUND: Cell-free DNA is a DNA fragment that is produced by cell apoptosis which can affect the micro-environment of cell apoptosis. The levels of Cell-free DNA have been associated with successful rate of in vitro fertilization-embryo transfer (IVF-ET) and embryonic development. Our aim is to determine the relationship between cell-free DNA and embryo quality. The mechanisms of cell-free DNA in granulose and the apoptosis will be determined also. METHODS: The study enrolled patients who were undergone IVF for the first time and grouped the patients as pregnant (n=130) and non-pregnant (n=59). The relationship was determined by statistical analysis between the levels of cell-free DNA in the follicular fluid and clinical data of IVF patients. Flow cytometry was done to detect the rate of granulosa cell apoptosis and intracellular reactive oxygen species (ROS) level. Western blotting and fluorescent quantitative PCR detected the apoptosis-related gene expressions. RESULTS: Clinical data statistics showed that cell-free DNA levels were positively correlated with granulosa cell apoptosis and negatively correlated with embryo quality and pregnancy rates. High levels of cell-free DNA lead to increased ROS in granulosa cells and activated caspase through Fas/FasL that induced apoptosis. CONCLUSION: High levels of cell-free DNA triggers granulosa cell apoptosis and influences oocyte maturation embryo development and pregnancy rates in IVF treatments. Cell-free DNA can be as a secondary criteria and predictive marker for the quality control of IVF embryo.