Early DNase-I therapy delays secondary brain damage after traumatic brain injury in adult mice.

Traumatic brain injury (TBI) causes the release of danger-associated molecular patterns (DAMP) from damaged or dead cells, which contribute to secondary brain damage after TBI. Cell-free DNA (cfDNA) is a DAMP known to cause disruption of the blood-brain barrier (BBB), promote procoagulant processes, brain edema, and neuroinflammation. This study tested the hypothesis that administration of deoxyribonuclease-I (DNase-I) has a beneficial effect after TBI. Mice (n = 84) were subjected to controlled cortical impact (CCI) and posttraumatic intraperitoneal injections of low dose (LD) or high dose (HD) of DNase-I or vehicle solution at 30 min and 12 h after CCI. LD was most effective to reduce lesion volume (p = 0.003), brain water content (p < 0.0001) and to stabilize BBB integrity (p = 0.019) 1 day post-injury (dpi). At 6 h post injury LD-treated animals showed less cleavage of fibrin (p = 0.0014), and enhanced perfusion as assessed by micro-computer-tomography (p = 0.027). At 5 dpi the number of Iba1-positive cells (p = 0.037) were reduced, but the number of CD45-positive cells, motoric function and brain lesion volume was not different. Posttraumatic-treatment with DNase-I therefore stabilizes the BBB, reduces the formation of brain edema, immune response, and delays secondary brain damage. DNase-I might be a new approach to extend the treatment window after TBI.

Retrieval of vector integration sites from cell-free DNA.

Gene therapy (GT) has rapidly attracted renewed interest as a treatment for otherwise incurable diseases, with several GT products already on the market and many more entering clinical testing for selected indications. Clonal tracking techniques based on vector integration enable monitoring of the fate of engineered cells in the blood of patients receiving GT and allow assessment of the safety and efficacy of these procedures. However, owing to the limited number of cells that can be tested and the impracticality of studying cells residing in peripheral organs without performing invasive biopsies, this approach provides only a partial snapshot of the clonal repertoire and dynamics of genetically modified cells and reduces the predictive power as a safety readout. In this study, we developed liquid biopsy integration site sequencing, or LiBIS-seq, a polymerase chain reaction technique optimized to quantitatively retrieve vector integration sites from cell-free DNA released into the bloodstream by dying cells residing in several tissues. This approach enabled longitudinal monitoring of in vivo liver-directed GT and clonal tracking in patients receiving hematopoietic stem cell GT, improving our understanding of the clonal composition and turnover of genetically modified cells in solid tissues and, in contrast to conventional analyses based only on circulating blood cells, enabling earlier detection of vector-marked clones that are aberrantly expanding in peripheral tissues.

Topical nanoparticles interfering with the DNA-LL37 complex to alleviate psoriatic inflammation in mice and monkeys.

Cell-free DNA (cfDNA) released from damaged or dead cells combines with LL37 and is converted into an immune response activator to exacerbate psoriasis. Here, we show that cationic nanoparticles (cNPs) efficiently compete for DNA from the DNA-LL37 immunocomplex and inhibit DNA-LL37-induced cell activation. Using phenotypical images, psoriasis area and severity index scoring, histology, and immunohistochemical analysis, we demonstrate that topical application of cNPs on psoriasiform skin of a mouse model relieves psoriatic symptoms. It is noteworthy that the results were confirmed in a cynomolgus monkey model. Moreover, topically administrated cNPs showed low in vivo toxicity because of their retention in skin. Mechanistic analyses of cytokine expression in the psoriatic site, cfDNA levels in circulation and inflamed skin, skin permeation, and biodistribution of cNPs also matched the therapeutic outcomes. Therefore, we present a previously unidentified strategy of nanomedicine to treat skin inflammatory diseases, which demonstrates great potential for clinical application.

Pro-inflammatory role of cell-free mitochondrial DNA in cardiovascular diseases.

Cardiovascular disease (CVD) is a major cause of morbidity and mortality. Inflammation contributes to the pathogenesis and progression of CVD. Circulating cell-free mitochondrial DNA (ccf-mtDNA) is that mtDNA fragments are released outside the cell and into the circulation by cell necrosis and secretion. The levels of ccf-mtDNA are increased in CVD and associated risk conditions, including hypercholesterolemia, diabetes mellitus, and arterial hypertension. MtDNA containing unmethylated CpG dinucleotides and can trigger inflammation that aggravates tissue injury by activating toll-like receptor 9, inflammasomes, and the stimulator of interferon genes pathway. Here, we review the expanding field of ccf-mtDNA-mediated inflammation and its role in the progression of CVD.

Tuned Cationic Dendronized Polymer: Molecular Scavenger for Rheumatoid Arthritis Treatment.

Cell-free deoxyribonucleic acid (cfDNA) released from either dead or damaged cells serves as a key autoantigen in rheumatoid arthritis (RA). They can be recognized by nucleic acid (NA) sensors such as the toll-like receptor (TLR), leading to activation of the innate immune system and chronic inflammation. Developed here is a cationic molecular scavenger, by screening cationic dendronized polymers, which eliminates cfDNA and inhibits TLR recognition and nucleic-acid-induced inflammation. The structure-property study demonstrates that toxicity, NA binding capacity, and biodistribution could be balanced to achieve maximum therapeutic effect by exquisite control of the molecular structure. In addition, the optimized cationic polymer effectively inhibited joint swelling, synovial hyperplasia, and bone destruction in collagen-induced arthritis (CIA) rat models. The results offer support for synthetic polymers offering new paradigm in autoimmune disease treatment.

Cationic nanoparticle as an inhibitor of cell-free DNA-induced inflammation.

Cell-free DNA (cfDNA) released from damaged or dead cells can activate DNA sensors that exacerbate the pathogenesis of rheumatoid arthritis (RA). Here we show that ~40 nm cationic nanoparticles (cNP) can scavenge cfDNA derived from RA patients and inhibit the activation of primary synovial fluid monocytes and fibroblast-like synoviocytes. Using clinical scoring, micro-CT images, MRI, and histology, we show that intravenous injection of cNP into a CpG-induced mouse model or collagen-induced arthritis rat model can relieve RA symptoms including ankle and tissue swelling, and bone and cartilage damage. This culminates in the manifestation of partial mobility recovery of the treated rats in a rotational cage test. Mechanistic studies on intracellular trafficking and biodistribution of cNP, as well as measurement of cytokine expression in the joints and cfDNA levels in systemic circulation and inflamed joints also correlate with therapeutic outcomes. This work suggests a new direction of nanomedicine in treating inflammatory diseases.

Circulating cell-free DNA as a biomarker in the diagnosis and prognosis of colorectal cancer

Abstract Colorectal cancer (CRC) is a disease without evident clinical symptoms in early stages, leading to late diagnosis and disease management. Current diagnostic and prognostic tools require invasive procedures and circulating molecular biomarkers fail to have optimal sensitivity and specificity. Circulating biomarkers with high clinical performance may be valuable for early diagnosis and prognosis of CRC. The purpose of this review was to investigate the application of circulating cell-free DNA (ccfDNA) in CRC diagnosis and prognosis and the analytical methods used in blood samples in articles published between 2005 and 2016. Based on specific inclusion and exclusion criteria, 26 articles were selected. Most studies used ccfDNA quantification as the molecular biomarker. The analytical method was mainly based on the quantitative polymerase chain reaction (qPCR). Biomarkers based on aberrantly methylated genes (n=6) and ccfDNA integrity/fragmentation (n=2) were also used for the CRC diagnosis. The CRC prognosis used the detection of oncogene mutations, such as KRAS and BRAF, in ccfDNA. Significant differences were found in variables among the studies revealing potential bias. ccfDNA quantification as a diagnostic biomarker for CRC has promising results but it lacks clinical specificity since other diseases present a similar increase in ccfDNA content. However, increasing research in the epigenomic field can lead the way to a clinically specific biomarker for the CRC early diagnosis. As for the analytical method, qPCR and derivatives seem to be a perfectly valid technique. The use of ccfDNA quantification in CRC prognosis seems promising. The attempt to use the ccfDNA quantification in clinical practice may reside in the prognosis using a qPCR technique.