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2022 ◽  
Vol 14 (1) ◽  
Author(s):  
Jinpeng Sun ◽  
Gang Wang ◽  
Haoran Wang ◽  
Feng Hua ◽  
Zeyuan Song ◽  
...  

AbstractExcessive epidural fibrosis attached to the dura mater is the major cause of recurrent failed back surgery syndrome after spine surgery. Neutrophil extracellular traps (NETs) promote epidural fibrosis, raising the possibility that the DNA backbone of NETs may be a potential target in the therapy of epidural fibrosis. Human body temperature-sensitive hydroxypropyl chitin hydrogel solutions were prepared to encapsulate DNase I, which gradually decomposed in vivo. DNase I, which was released from temperature-sensitive hydrogels, destroyed the DNA backbone of NETs and dispersed the clustering of myeloperoxidase (MPO) in NETs. Evidence from MRI, H&E and Masson staining supported that hydroxypropyl chitin hydrogels loaded with DNase I were nontoxic and reduced epidural fibrosis. As expected, fibronectin in the wound was significantly abridged in the mice treated with hydrogels loaded with DNase I. Compared with the gelatin sponge absorbing DNase I, temperature-sensitive hydroxypropyl chitin hydrogels loaded with DNase I were more powerful in the therapy of epidural fibrosis. These results indicate that temperature-sensitive hydroxypropyl chitin hydrogels were effective in DNase I encapsulation and alleviation of epidural fibrosis in a mouse model of laminectomy.


Cells ◽  
2022 ◽  
Vol 11 (2) ◽  
pp. 192
Author(s):  
Moritz Lenz ◽  
Thomas Maiberger ◽  
Lina Armbrust ◽  
Antonia Kiwit ◽  
Axel Von der Wense ◽  
...  

Introduction: An early and accurate diagnosis of early onset neonatal sepsis (EONS) and late onset neonatal sepsis (LONS) is essential to improve the outcome of this devastating conditions. Especially, preterm infants are at risk. Reliable biomarkers are rare, clinical decision-making depends on clinical appearance and multiple laboratory findings. Markers of NET formation and NET turnover might improve diagnostic precision. Aim of this study was to evaluate the diagnostic value of NETs in sepsis diagnosis in neonatal preterm infants. Methods: Plasma samples of neonatal preterm infants with suspected sepsis were collected. Blood samples were assayed for markers of NET formation and NET turnover: cfDNA, DNase1, nucleosome, NE, and H3Cit. All clinical findings, values of laboratory markers, and epidemiological characteristics were collected retrospectively. Two subpopulations were created to divide EONS from LONS. EMA sepsis criteria for neonatal sepsis were used to generate a sepsis group (EMA positive) and a control group (EMA negative). Results: A total of 31 preterm neonates with suspected sepsis were included. Out of these, nine patients met the criteria for sepsis according to EMA. Regarding early onset neonatal sepsis (3 EONS vs. 10 controls), cfDNA, DNase I, nucleosome, and CRP were elevated significantly. H3Cit and NE did not show any significant elevations. In the late onset sepsis collective (6 LONS vs. 12 controls), cfDNA, DNase I, and CRP differed significantly compared to control group.


2021 ◽  
Vol Volume 16 ◽  
pp. 8121-8138
Author(s):  
Jia-You Fang ◽  
Wei-Ling Chou ◽  
Chwan-Fwu Lin ◽  
Calvin T Sung ◽  
Ahmed Alalaiwe ◽  
...  

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Grigorios Georgolopoulos ◽  
Nikoletta Psatha ◽  
Mineo Iwata ◽  
Andrew Nishida ◽  
Tannishtha Som ◽  
...  

AbstractLineage commitment and differentiation is driven by the concerted action of master transcriptional regulators at their target chromatin sites. Multiple efforts have characterized the key transcription factors (TFs) that determine the various hematopoietic lineages. However, the temporal interactions between individual TFs and their chromatin targets during differentiation and how these interactions dictate lineage commitment remains poorly understood. Here we perform dense, daily, temporal profiling of chromatin accessibility (DNase I-seq) and gene expression changes (total RNA-seq) along ex vivo human erythropoiesis to comprehensively define developmentally regulated DNase I hypersensitive sites (DHSs) and transcripts. We link both distal DHSs to their target gene promoters and individual TFs to their target DHSs, revealing that the regulatory landscape is organized in distinct sequential regulatory modules that regulate lineage restriction and maturation. Finally, direct comparison of transcriptional dynamics (bulk and single-cell) and lineage potential between erythropoiesis and megakaryopoiesis uncovers differential fate commitment dynamics between the two lineages as they exit the stem and progenitor stage. Collectively, these data provide insights into the temporally regulated synergy of the cis- and the trans-regulatory components underlying hematopoietic lineage commitment and differentiation.


Acta Naturae ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 15-23
Author(s):  
Dmitry V. Volkov ◽  
George V. Tetz ◽  
Yury P. Rubtsov ◽  
Alexey V. Stepanov ◽  
Alexander G. Gabibov

Antitumor therapy, including adoptive immunotherapy, inevitably faces powerful counteraction from advanced cancer. If hematological malignancies are currently amenable to therapy with CAR-T lymphocytes (T-cells modified by the chimeric antigen receptor), solid tumors, unfortunately, show a significantly higher degree of resistance to this type of therapy. As recent studies show, the leading role in the escape of solid tumors from the cytotoxic activity of immune cells belongs to the tumor microenvironment (TME). TME consists of several types of cells, including neutrophils, the most numerous cells of the immune system. Recent studies show that the development of the tumor and its ability to metastasize directly affect the extracellular traps of neutrophils (neutrophil extracellular traps, NETs) formed as a result of the response to tumor stimuli. In addition, the nuclear DNA of neutrophils the main component of NETs erects a spatial barrier to the interaction of CAR-T with tumor cells. Previous studies have demonstrated the promising potential of deoxyribonuclease I (DNase I) in the destruction of NETs. In this regard, the use of eukaryotic deoxyribonuclease I (DNase I) is promising in the effort to increase the efficiency of CAR-T by reducing the NETs influence in TME. We will examine the role of NETs in TME and the various approaches in the effort to reduce the effect of NETs on a tumor.


2021 ◽  
Vol 22 (21) ◽  
pp. 12074
Author(s):  
Ludmila Alekseeva ◽  
Aleksandra Sen’kova ◽  
Innokenty Savin ◽  
Marina Zenkova ◽  
Nadezhda Mironova

Tumor-associated cell-free DNAs (cfDNA) play an important role in the promotion of metastases. Previous studies proved the high antimetastatic potential of bovine pancreatic DNase I and identified short interspersed nuclear elements (SINEs) and long interspersed nuclear elements (LINEs)and fragments of oncogenes in cfDNA as the main molecular targets of enzyme in the bloodstream. Here, recombinant human DNase I (commercial name Pulmozyme®), which is used for the treatment of cystic fibrosis in humans, was repurposed for the inhibition of lung metastases in the B16 melanoma model in mice. We found that Pulmozyme® strongly reduced migration and induced apoptosis of B16 cells in vitro and effectively inhibited metastases in lungs and liver in vivo. Pulmozyme® was shown to be two times more effective when administered intranasally (i.n.) than bovine DNase I, but intramuscular (i.m.) administration forced it to exhibit as high an antimetastatic activity as bovine DNase I. Both DNases administered to mice either i.m. or i.n. enhanced the DNase activity of blood serum to the level of healthy animals, significantly decreased cfDNA concentrations, efficiently degraded SINE and LINE repeats and c-Myc fragments in the bloodstream and induced apoptosis and disintegration of neutrophil extracellular traps in metastatic foci; as a result, this manifested as the inhibition of metastases spread. Thus, Pulmozyme®, which is already an approved drug, can be recommended for use in the treatment of lung metastases.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2096-2096
Author(s):  
Anh T.P. Ngo ◽  
Irene Yarovoi ◽  
Guohua Zhao ◽  
Amrita Sarkar ◽  
Lubica Rauova ◽  
...  

Abstract Sepsis is a dysregulated response to infection leading to life-threating organ damage. Although it remains one of the most common causes of mortality worldwide, it lacks targeted treatments. Neutrophils play a crucial role in sepsis by releasing NETs, webs of DNA complexed with histones and antimicrobial proteins that capture pathogens and prevent bacterial dissemination. However, when NETs are degraded by circulating nucleases they release NET-degradation products (NDPs) including cell-free (cf) DNA, histones and myeloperoxidase, which trigger coagulation, induce complement activation, and cause oxidative tissue damage. We proposed a novel NET-directed therapy in sepsis, in which NETs are stabilized by the platelet chemokine PF4. Binding of PF4 enhances NET DNase-resistance, promotes NDP sequestration and increases bacterial capture, improving survival in murine sepsis. As NETs are considered prothrombotic, we were concerned that NET stabilization may increase the risk of clot formation. We therefore sought to determine the effect of PF4-NET stabilization on the thrombogenicity of NETs to learn if this strategy is safe for clinical application. To that end, we examined the effect of PF4 on the thrombotic potential of DNA and NET fragments at different states of nuclease digestion. High molecular weight (hmw) genomic DNA (hmwDNA, >50kbp) was isolated from human whole blood. hmwDNA was digested with restriction enzymes (EcoRI and AluI) for 15min to generate DNA fragments of ~4kbp and ~250bp, respectively. Neutrophils were also isolated from human blood and stimulated with 100 nM PMA to produce neutrophil-adherent NETs, which were cleaved from cell bodies by treatment with 4U/mL DNase I for 20 minutes, releasing NETs >50kbp (hmwNETs). Additional incubation of hmwNETs with DNase I yielded smaller NET fragments. We assessed in vitro activation of coagulation by DNA and NETs by measuring thrombin generation and fibrin formation in platelet-poor plasma using fluorogenic substrate and turbidity assays. Neutrophil-adherent NETs induced far less thrombin generation and fibrin formation in plasma than hmwDNA and hmwNETs. PF4 significantly increased lag time and reduced peak thrombin formation induced by both hmwDNA and hmwNETs. Binding of PF4 also delayed clot initiation time and reduced the rate of fibrin generation. Digestion of hmwDNA and hmwNETs to smaller fragments markedly enhanced thrombogenicity. We posited that shorter DNA fragments are more thrombogenic because they have a greater proportion of end-fragment DNA that exposes more single-stranded DNA. To test this hypothesis, we subjected hmwDNA and digested DNA to heat denaturation at 95°C and rapid cooling to generate single stranded DNA and found that this accelerated fibrin generation. Although the anti-thrombotic effect of PF4 was most pronounced with longer DNA and NET fragments, it continued to significantly reduce fibrin generation induced by shorter DNA fragments, perhaps by stabilizing the fragments to prevent exposure of single-stranded DNA. In conclusion, although prior studies have shown that NETs increase the risk of thrombosis in sepsis, we propose the counter-intuitive concept that PF4-stabilization decreases the risk of NET-mediated prothrombotic state by (1) inhibiting DNase cleavage of intact NETs and subsequent liberation of prothrombotic cfDNA from non-thrombogenic neutrophil-adherent NETs, and (2) preventing further digestion of circulating cfDNA into shorter and more prothrombotic fragments. Although NETs are a double-edged sword: capable of capturing pathogens but inducing host-tissue damage and thrombosis when degraded, treatment with PF4 tips the balance, limiting the capacity of NETs to induce fibrin generation and thrombosis, while enhancing their ability to fight infection by microbial entrapment. These studies add support to our hypothesis that PF4 stabilization of NETs is protective in sepsis and merits further investigation in translational studies. Disclosures No relevant conflicts of interest to declare.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1020-1020
Author(s):  
Noritaka Yada ◽  
Jingrui Sui ◽  
Liang Zheng ◽  
X. Long Zheng

Abstract Introduction. Immune thrombotic thrombocytopenic purpura (iTTP), a potentially fatal blood disorder, is primarily caused by severe deficiency of plasma ADAMTS13 activity resulting from immunoglobulin (Ig) G-mediated inhibition of plasma ADAMTS13 activity. However, severe ADAMTS13 deficiency is necessary but not sufficient to cause acute iTTP. An environmental factor such as infection or acute inflammation may be necessary to trigger the acute onset of the disease. We and others have previously reported that plasma markers of neutrophil activation and neutrophil extracellular traps (NETs) formation are significantly elevated in patients with acute iTTP, which returns to normal during remission. However, the pathogenetic role of NETs in acute iTTP is not fully understood. Methods and results. Using flow cytometry, microfluidic shear-based assay, and confocal imaging analysis, we determined the in vivo NETosis in blood samples obtained from patients with acute episode of iTTP and ex vivo NETs formation, as well as the therapeutic efficacy of DNase I on thrombus formation under flow. We showed that by flow cytometry that only very few CitH3+/MPO+ positive neutrophils were present in the healthy donor blood. This population of cells dramatically increased after being stimulated with a bacterial toxin (i.e., Shigatoxin-2) at ~100 ng/mL for 15 min. Importantly, the number of CitH3+/MPO+ positive neutrophils in the sample obtained from a patient with acute iTTP was ~1,000 times higher than that in the healthy controls (Fig. 1), suggesting a massive NETosis in patients with acute iTTP. Microfluidic shear-based assay and confocal imaging analysis further confirmed a dramatic increase in adhesion and aggregation of murine platelets (stained with Alexa647 anti-CD41) and neutrophil (stained with Hoechst), as well as formation of NETs (stained with Syto green) following a perfusion of an Adamts13 -/- murine whole blood (anti-coagulated with thrombin inhibitor, PPACK) under arterial shear (15 dyne/cm 2) over a stimulated murine endothelial surface. Interestingly, an addition of DNase I (100 U/mL) significantly reduced the overall surface coverage of platelets and neutrophils on the murine endothelial surface under the same conditions (Fig. 2). Conclusions. These results demonstrate for the first time NETosis and NETs formation are common in patients with acute iTTP and in Adamts13 -/- mice after being stimulated with shigatoxin; DNase I appears to be highly efficacious eliminating the NETs and platelet/neutrophil-dominant thrombosis under arterial flow. Our findings support the pathogenetic role of NETs in the onset and progression of iTTP, and the therapeutic potential of DNase I in such a fatal disease. Figure 1 Figure 1. Disclosures Zheng: Alexion: Speakers Bureau; Sanofi-Genzyme: Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria; Clotsolution: Other: Co-founder; AJMC: Honoraria.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hamish C. L. Yau ◽  
Adam K. Malekpour ◽  
Nazarmohammad G. Momin ◽  
Ana L. Morales-García ◽  
William G. T. Willats ◽  
...  

AbstractWashed textiles can remain malodorous and dingy due to the recalcitrance of soils. Recent work has found that ‘invisible’ soils such as microbial extracellular DNA (eDNA) play a key role in the adhesion of extracellular polymeric substances that form matrixes contributing to these undesirable characteristics. Here we report the application of an immunostaining method to illustrate the cleaning mechanism of a nuclease (DNase I) acting upon eDNA. Extending previous work that established a key role for eDNA in anchoring these soil matrixes, this work provides new insights into the presence and effective removal of eDNA deposited on fabrics using high-resolution in-situ imaging. Using a monoclonal antibody specific to Z-DNA, we showed that when fabrics are washed with DNase I, the incidence of microbial eDNA is reduced. As well as a quantitative reduction in microbial eDNA, the deep cleaning benefits of this enzyme are shown using confocal microscopy and imaging analysis of T-shirt fibers. To the best of our knowledge, this is the first time the use of a molecular probe has been leveraged for fabric and homecare-related R&D to visualize eDNA and evaluate its removal from textiles by a new-to-laundry DNase enzyme. The approaches described in the current work also have scope for re-application to identify further cleaning technology.


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