A novel DNase assay reveals low DNase activity in severe asthma.

Secreted deoxyribonucleases (DNases), such as DNase-I and DNase-IL3, degrade extracellular DNA, and endogenous DNases have roles in resolving airway inflammation and guarding against autoimmune responses to nucleotides. Subsets of patients with asthma have high airway DNA levels, but information about DNase activity in health and in asthma is lacking. To characterize DNase activity in health and in asthma, we developed a novel kinetic assay using a Taqman probe sequence that is quickly cleaved by DNase-I to produce a large product signal. We used this kinetic assay to measure DNase activity in sputum from participants in the Severe Asthma Research Program (SARP)-3 (n = 439) and from healthy controls (n = 89). We found that DNase activity was lower than normal in asthma [78.7 relative fluorescence units (RFU)/min vs. 120.4 RFU/min, P < 0.0001]. Compared to patients with asthma with sputum DNase activity in the upper tertile activity levels, those in the lower tertile of sputum DNase activity were characterized clinically by more severe disease and pathologically by airway eosinophilia and airway mucus plugging. Carbamylation of DNase-I, a post-translational modification that can be mediated by eosinophil peroxidase, inactivated DNase-I. In summary, a Taqman probe-based DNase activity assay uncovers low DNase activity in the asthma airway that is associated with more severe disease and airway mucus plugging and may be caused, at least in part, by eosinophil-mediated carbamylation.NEW & NOTEWORTHY We developed a new DNase assay and used it to show that DNase activity is impaired in asthma airways.

Neutrophil Extracellular Trap Formation and Deoxyribonuclease I Activity in Patients with Otitis Media with Antineutrophil Cytoplasmic Antibody-Associated Vasculitis.

INTRODUCTION: No previous studies have evaluated the levels of neutrophil extracellular trap (NET) remnants or the importance of deoxyribonuclease (DNase) I activity based on the disease activity of otitis media with antineutrophil cytoplasmic antibody-associated vasculitis (OMAAV). The aim of this study was to explore the formation of NETs in the middle ear of patients with OMAAV during the onset and remission phases of the disease, with a particular focus on the relationships between the quantifiable levels of NET remnants and DNase I activity. METHODS: OMAAV patients were eligible for inclusion. Patients with otitis media with effusion (OME) were examined as controls. The levels of cell-free deoxyribonucleic acid (DNA), citrullinated-histone H3 (cit-H3)-DNA complex, and myeloperoxidase (MPO)-DNA complex were quantified using an enzyme-linked immunosorbent assay. DNase I activity was measured using a fluorometric method. RESULTS: The quantifiable levels of cell-free DNA, cit-H3-DNA complex, and MPO-DNA complex in the middle ear lavage of patients with OMAAV at onset were significantly higher than those in patients with OMAAV at remission and in patients with OME. DNase I activity in the patients with OMAAV at onset was significantly lower than those in patients with OMAAV at remission and OME and was negatively correlated with the level of MPO-DNA complex. CONCLUSIONS: This study suggests that NET remnants and DNase I activity may be potentially useful biomarkers for the diagnosis and disease activity of OMAAV.

Revising the value of Antistreptolysin O titre in childhood and its interpretation in the diagnostic approach of rheumatic diseases.

The burden of group A streptococcus (GAS) infection and its rheumatic sequelae remains dramatically high, especially in low-income countries. Recently, an increased number of Acute Rheumatic Fever (ARF) cases was documented in many regions of Italy. The diagnosis of rheumatic sequelae relies on clinical signs and on the evaluation of the Antistreptolysin O titre (ASO), whose variations are globally reported. To re-examine the standard reference value of ASO titre, by measuring either its upper limit of normal (ULN) in a population of healthy children (HC) or comparing these values with streptococcal antibodies registered in a cohort of patients affected by the rheumatic sequelae of GAS infection. We performed a multicenter retrospective study. We enrolled 125 HC, aged 2-17 years, and a total of 181 patients affected by ARF, acute streptococcal pharyngitis, post-streptococcal arthritis, Henoch-Schönlein purpura and erythema nodosum, divided into four groups. The levels of ASO and anti-deoxyribonuclease B (anti-DNase B) titres were analyzed and compared among the various groups. Moreover, the 80th percentile value was calculated and established as the ULN for ASO titre in HC group. The ULN for ASO titre in overall HC group was 515 IU/mL, resulting in higher than used in the routine investigation. The ASO titre was significantly higher in patients with rheumatic sequelae compared with HC group, with a peak in the age between 5 and 15 years.    Conclusion: Our study established a new ULN normal value of streptococcal serology in a childhood and adolescent population of Italy, suggesting the need to extend this revaluation to the critical areas, in order to avoid underestimating ARF diagnosis. The correct interpretation of ASO and anti-DNase B values in the context of rheumatic diseases has been discussed. What is Known: • The global burden of disease caused by group A streptococcus is not known and remains an important cause of morbidity and mortality. Acute rheumatic fever continues to be a serious worldwide public health problem and a recent recurrence of group A streptococcus infection cases is observed. • The streptococcal sequelae requires evidence of preceding streptococcal infection, commonly elevated streptococcal antibody titre, but the upper limit for these titres varies considerably based on age group, region, and origin. What is New: • This study provides population-specific values for streptococcal antibody titres in Italy. • Interpret the results of group A streptococcal antibody tests within the clinical context.

Reactivation of nucleases with peroxidation damages induced by a menadione: ascorbate combination devastates human prostate carcinomas: ultrastructural aspects.

INTRODUCTION: Xenografts of androgen-independent human DU145 prostate metastatic carcinomas implanted in nu/nu male mice have revealed a significant survival after a prooxidant anticancer treatment consisting of a combination of menadione bisulfite and sodium ascorbate (VK3:VC). METHODS: Implanted samples of diaphragm carcinomas from longest survived mice from either oral, intraperitoneal (IP), or both oral and IP treatment groups were assessed with light, scanning, and transmission electron microscopy to analyze morphologic damages. RESULTS: Compared with previous fine structure data of in vitro untreated carcinomas, the changes induced by oral, IP, and oral with IP VK3:VC treatment dismantled those xenografts with autoschizis, and necrotic atrophy was accomplished by cell's oxidative stress whose injuries were consequent to reactivated deoxyribonucleases and ribonucleases. Tumor destructions resulted from irreversible damages of nucleus components, endoplasmic reticulum, and mitochondria there. Other alterations included those of the cytoskeleton that resulted in characteristic self-excisions named " autoschizis." All these injuries lead resilient cancer cells to necrotic cell death. CONCLUSION: The fine structure damages caused by VK3:VC prooxidant combination in the human DU145 prostate xenografts confirmed those shown in vitro and of other cell lines with histochemistry and biomolecular investigations. These devastations incurred without damage to normal tissues; thus, our data brought support for the above combination to assist in the treatment of prostate cancers and other cancers.

Variability of endogenous deoxyribonuclease activity and its pathophysiological consequences.

Deoxyribonucleases (DNases) are enzymes that cleave DNA. Some DNases are secreted outside of cells where they can cleave extracellular DNA (ecDNA). High concentration of ecDNA is associated with diseases such as sepsis, preeclampsia, and systemic lupus. DNA can be released from dying cells and is immunogenic. DNases cleave ecDNA and might prevent activation of the immune system. Low DNase activity could be disadvantageous in diseases where high amounts of ecDNA are released from dying cells. The relationship between DNase activity and ecDNA remains unknown. The lack of standard values in DNase activity makes the studies difficult to compare. This review focuses on summarizing methods for DNase activity measurements, the possible implication of decreased DNase activity in diseases, and the impact on diseases associated with a high concentration of ecDNA.

Zinc oxide nanoparticles (ZnO-NPs) exhibit immune toxicity to crucian carp (Carassius carassius) by neutrophil extracellular traps (NETs) release and oxidative stress.

Zinc oxide nanoparticles (ZnO-NPs) are widely used in sunscreens, cosmetics, paint, construction materials, and other products. ZnO-NPs released into the environment can harm aquatic creatures and pose a health risk to humans through the food chain. ZnO-NPs are toxic to fish, but there are few reports on its immunotoxicity on crucian carp (Carassius carassius). In this study, ZnO-NPs increased the biochemical indexes of the liver in serum, including aspartate aminotransferase (AST) and alanine aminotransferase (ALT). In histopathological observation, many inflammatory cells were filled in the liver's central vein stimulated by ZnO-NPs. Furthermore, ZnO-NPs could increase malondialdehyde (MDA) level, lessen superoxide dismutase (SOD) level, and elevate the level of neutrophil extracellular traps (NETs). However, deoxyribonuclease I (DNase I) alleviated all biochemical indexes and histopathological changes. Immunofluorescence in vitro confirmed that NETs were composed of citrullinated histone 3, myeloperoxidase, and neutrophil elastase. ZnO-NPs-increased NETs were dependent on reactive oxygen species (ROS) and nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase and were also related to partial processes of glycolysis. Our study confirms that ZnO-NPS has a toxic effect on the liver of crucian carp. DNase I can prevent liver damage caused by ZnO-NPs, which provides a new insight into the immunotoxicity of ZnO-NPs to fish.

Alteplase Dose Assessment for Pleural infection Therapy (ADAPT) Study-2: Use of 2.5 mg alteplase as a starting intrapleural dose.

BACKGROUND AND OBJECTIVE: Intrapleural tissue plasminogen activator/deoxyribonuclease (tPA/DNase) therapy is increasingly used in pleural infection. Bleeding risks and costs associated with tPA remain the clinical concerns. Our dose de-escalation series aims to establish the lowest effective dosing regimen for tPA/DNase. This study assesses the intrapleural use of 2.5 mg tPA/5 mg DNase for pleural infection. METHODS: Consecutive patients with pleural infection treated with a starting regime of 2.5 mg tPA/5 mg DNase were included from two centres in Australia and UK. Escalation of tPA dose was permitted if clinical response was inadequate. RESULTS: Sixty-nine patients (mean age 61.0 years) received intrapleural 2.5 mg tPA/5 mg DNase. Most (88.4%) were treated successfully and discharged from hospital without surgery by 90 days. Patients received a median of 5 [interquartile range [IQR] = 3-6] doses of tPA/DNase. Total amount of tPA used per patient was 12.5 mg [median, IQR = 7.5-15.0]. Seventeen patients required dose escalation of tPA; most (n = 12) for attempted drainage of distant non-communicating locule(s). Treatment success was corroborated by clearance of pleural opacities on radiographs (from median 27.0% [IQR = 17.1-44.5] to 11.0% [IQR = 6.4-23.3] of hemithorax, p < 0.0001), increased pleural fluid drainage (1.98 L [median, IQR = 1.38-2.68] over 72 h following commencement of tPA/DNase) and reduction of serum C-reactive protein level (by 45.0% [IQR = 39.3-77.0] from baseline at day 5, p < 0.0001). Two patients required surgery. Six patients with significant comorbidities (e.g., advanced cancer) had ongoing infection when palliated and died. Two patients experienced self-limiting pleural bleeding and received blood transfusion. CONCLUSION: A starting intrapleural regime of 2.5 mg tPA/5 mg DNase, with up-titration if needed, can be effective and deserves further exploration.

Neutrophil Extracellular Traps in Atherosclerosis and Thrombosis.

Despite effective therapeutic and preventive strategies, atherosclerosis and its complications still represent a substantial health burden. Leukocytes and inflammatory mechanisms are increasingly recognized as drivers of atherosclerosis. Neutrophil granulocytes within the circulation were recently shown to undergo neutrophil extracellular trap (NET) formation, linking innate immunity with acute complications of atherosclerosis. In this chapter, we summarize mechanisms of NET formation, evidence for their involvement in atherosclerosis and thrombosis, and potential therapeutic regimens specifically targeting NET components.

Processing and integration of functionally oriented prespacers in the Escherichia coli CRISPR system depends on bacterial host exonucleases.

CRISPR-Cas systems provide bacteria with adaptive immunity against viruses. During spacer adaptation, the Cas1-Cas2 complex selects fragments of foreign DNA, called prespacers, and integrates them into CRISPR arrays in an orientation that provides functional immunity. Cas4 is involved in both the trimming of prespacers and the cleavage of protospacer adjacent motif (PAM) in several type I CRISPR-Cas systems, but how the prespacers are processed in systems lacking Cas4, such as the type I-E and I-F systems, is not understood. In Escherichia coli, which has a type I-E system, Cas1-Cas2 preferentially selects prespacers with 3' overhangs via specific recognition of a PAM, but how these prespacers are integrated in a functional orientation in the absence of Cas4 is not known. Using a biochemical approach with purified proteins, as well as integration, prespacer protection, sequencing, and quantitative PCR assays, we show here that the bacterial 3'-5' exonucleases DnaQ and ExoT can trim long 3' overhangs of prespacers and promote integration in the correct orientation. We found that trimming by these exonucleases results in an asymmetric intermediate, because Cas1-Cas2 protects the PAM sequence, which helps to define spacer orientation. Our findings implicate the E. coli host 3'-5' exonucleases DnaQ and ExoT in spacer adaptation and reveal a mechanism by which spacer orientation is defined in E. coli.

CRISPR-Cas12a has widespread off-target and dsDNA-nicking effects.

Cas12a (Cpf1) is an RNA-guided endonuclease in the bacterial type V-A CRISPR-Cas anti-phage immune system that can be repurposed for genome editing. Cas12a can bind and cut dsDNA targets with high specificity in vivo, making it an ideal candidate for expanding the arsenal of enzymes used in precise genome editing. However, this reported high specificity contradicts Cas12a's natural role as an immune effector against rapidly evolving phages. Here, we employed high-throughput in vitro cleavage assays to determine and compare the native cleavage specificities and activities of three different natural Cas12a orthologs (FnCas12a, LbCas12a, and AsCas12a). Surprisingly, we observed pervasive sequence-specific nicking of randomized target libraries, with strong nicking of DNA sequences containing up to four mismatches in the Cas12a-targeted DNA-RNA hybrid sequences. We also found that these nicking and cleavage activities depend on mismatch type and position and vary with Cas12a ortholog and CRISPR RNA sequence. Our analysis further revealed robust nonspecific nicking of dsDNA when Cas12a is activated by binding to a target DNA. Together, our findings reveal that Cas12a has multiple nicking activities against dsDNA substrates and that these activities vary among different Cas12a orthologs.