Serum DNase Research Articles

Neutrophils release extracellular DNA traps in response to exercise.

Intense exercise evokes a rapid and transient increase in circulating cell-free DNA (cf-DNA), a phenomenon that is commonly observed in a variety of acute and chronic inflammatory conditions. In this study, we aimed to shed new light on the release and clearance mechanisms of cf-DNA in response to exercise. We hypothesized that activated neutrophils may primarily contribute to exercise-evoked cf-DNA levels by releasing neutrophil extracellular traps (NETs). Analysis of plasma and/or serum samples from male athletes at rest and in response to exhaustive treadmill exercise revealed an immediate and transient increase in cf-DNA that was concomitantly counterbalanced by an increase in serum DNase activity. Consistently, rapid release and clearance kinetics for cf-DNA could also be observed in response to intensive cycling exercise, with no significant differences between endurance-trained (V̇o2max >57 ml·min(-1)·kg(-1)) and healthy (V̇o2max <49 ml·min(-1)·kg(-1)) sedentary individuals. In postexercise blood smear samples, we detected seemingly intact neutrophils displaying morphological signs of NET release, as indicated by abnormal swollen nuclei and emanating DNA fibers. In support, we observed a striking correlation of postexercise cf-DNA concentrations with plasma levels of the granule-derived enzyme myeloperoxidase. Our study indicates that intense exercise induces liberation of NETs, which is sufficiently counterbalanced in healthy individuals by a concomitant rise in serum DNase activity. As aberrant release of NETs has been linked to diverse disease states, monitoring of cf-DNA/DNase levels or activities in response to standardized exercise testing could provide a valuable tool to identify people who are at increased risk for cardiac ischemia, thrombosis, autoimmunity, or chronic fatigue.

Serum-dependent processing of late apoptotic cells for enhanced efferocytosis.

Binding of the serum protein complement component C1q to the surface of dying cells facilitates their clearance by phagocytes in a process termed efferocytosis. Here, we investigate during which phase of apoptotic cell death progression C1q binding takes place. Purified C1q was found to bind to all dying cells and, albeit weaker, also to viable cells. The presence of serum abrogated completely the binding to viable cells. In addition, C1q binding to dying cells was limited to a specific subpopulation of late apoptotic/secondary necrotic cells. Co-culturing serum-treated apoptotic cells with human monocytes revealed a much higher phagocytosis of C1q-positive than of C1q-negative late apoptotic/secondary necrotic cells. But this phagocytosis-promoting activity could not be observed with purified C1q. Serum-treated C1q-positive late apoptotic/secondary necrotic cells exhibited a similar volume, a similar degraded protein composition, but a much lower DNA content in comparison with the remaining late apoptotic/secondary necrotic cells. This was mediated by a serum-bound nuclease activity that could be abrogated by G-actin, which is a specific inhibitor of serum DNase I. These results show that serum factors are involved in the prevention of C1q binding to viable cells and in the processing of late apoptotic/secondary necrotic cells promoting cell death progression toward apoptotic bodies. This process leads to the exposure of C1q-binding structures and facilitates efferocytosis.

Cooperation of Factor VII–Activating Protease and Serum DNase I in the Release of Nucleosomes From Necrotic Cells

Removal of dead cells is essential in the maintenance of tissue homeostasis, and efficient removal prevents exposure of intracellular content to the immune system, which could lead to autoimmunity. The plasma protease factor VII-activating protease (FSAP) can release nucleosomes from late apoptotic cells. FSAP circulates as an inactive single-chain protein, which is activated upon contact with either apoptotic cells or necrotic cells. The purpose of this study was to investigate the role of FSAP in the release of nucleosomes from necrotic cells. Necrotic Jurkat cells were incubated with serum, purified 2-chain FSAP, and/or DNase I. Nucleosome release was analyzed by flow cytometry, and agarose gel electrophoresis was performed to detect DNA breakdown. Incubation with serum released nucleosomes from necrotic cells. Incubation with FSAP-deficient serum or serum in which FSAP was inhibited by a blocking antibody was unable to release nucleosomes from necrotic cells, confirming that FSAP is indeed the essential serum factor in this process. Together with serum DNase I, FSAP induced the release of DNA from the cells, the appearance of nucleosomes in the supernatant, and the fragmentation of chromatin into eventually mononucleosomes. FSAP and DNase I are the essential serum factors that cooperate in necrotic cell DNA degradation and nucleosome release. We propose that this mechanism may be important in the removal of potential autoantigens.

FRI0023 Enzymatic dnase and hyaluronidase serum activity in patients with early arthritis

BackgroundEarly arthritis (EA) is one of the most important problems of modern rheumatology. Searching the new pathogenic mechanism of recently-onset joints inflammation caused by different factors is needed. Enzymatic serum...

Crystal Structure of a Voltage-gated K+ Channel Pore Module in a Closed State in Lipid Membranes

Voltage-gated K(+) channels underlie the electrical excitability of cells. Each subunit of the functional tetramer consists of the tandem fusion of two modules, an N-terminal voltage-sensor and a C-terminal pore. To investigate how sensor coupling to the pore generates voltage-dependent channel opening, we solved the crystal structure and characterized the function of a voltage-gated K(+) channel pore in a lipid membrane. The structure of a functional channel in a membrane environment at 3.1 Å resolution establishes an unprecedented connection between channel structure and function. The structure is unique in delineating an ion-occupied ready to conduct selectivity filter, a confined aqueous cavity, and a closed activation gate, embodying a dynamic entity trapped in an unstable closed state.

Serum DNase I activity in systemic lupus erythematosus: correlation with immunoserological markers, the disease activity and organ involvement

Decreased activity of serum desoxyribonuclease I (DNase I) in systemic lupus erythematosus (SLE) has been reported, but its role as a biomarker in SLE is still unelucidated. Seventy-seven SLE patients (aged 39.6 ± 13.1 years) were studied for serum DNase I activity, levels of antinuclear (ANA), anti-dsDNA [high-avidity ELISA, conventional ELISA and indirect immunofluorescence (IIF)], anti-nucleosome, anti-histone antibodies, complement components C3 and C4. SLE disease activity was evaluated by disease activity index (SLEDAI-2K). Thirty-five patients were serologically and clinically followed for 3-12 months (mean 5.6 ± 2.8). Thirty-seven healthy blood donors were the control group. DNase I activity in SLE patients was lower than in healthy controls (p<0.01). DNase I activity was in positive correlation with SLEDAI-2K (p<0.01), levels of ANA, anti-dsDNA, anti-nucleosome and anti-histone antibodies (p<0.01) and in negative correlation with C3 concentration (p<0.05). The highest correlation was found between DNase I activity and anti-dsDNA concentrations determined by high-avidity ELISA (r=0.624), followed by IIF (r=0.541) and conventional ELISA (r=0.405). In the follow-up study, DNase I activity also correlated with SLEDAI-2K (p<0.01). SLE patients with low DNase I activity more frequently had SLE-specific cutaneous lesions (p<0.05). Monitoring of DNase I activity simultaneously with SLEDAI-2K might be a useful tool in the follow-up of SLE. An increase of DNase I activity characterized relapse in most SLE patients, although it did not reach the levels of healthy individuals. A decrease of DNase I activity in SLE flare-ups might be a functional biomarker of a subset of patients with specific dysfunction of apoptotic chromatin degradation.

Genetic and expression analysis of SNPs in the human deoxyribonuclease II: SNPs in the promoter region reduce its in vivo activity through decreased promoter activity

Five SNPs in the human DNase II gene have been reported to be associated with rheumatoid arthritis (RA). Genotype and haplotype analysis of 14 SNPs, nine SNPs of which reported in the NCBI dbSNP database in addition to these five SNPs, was performed in healthy subjects. The enzymatic activities of the amino acid substituted DNase II corresponding to each SNP and serum DNase II in healthy Japanese, and promoter activities derived from each haplotype of the RA-related SNPs were measured. Significant correlations between genotype in each RA-related SNP and enzymatic activity levels were found; alleles associated with RA exhibited a reduction in serum DNase II activity. Furthermore, the promoter activities of each reporter construct corresponding to predominant haplotypes in three SNPs in the promoter region of the gene exhibited significant correlation with levels of serum DNase II activity. These findings indicate these three SNPs could alter the promoter activity of DNASE2, leading to a decline in DNase II activity in the serum through gene expression. Since the three SNPs in the promoter region of the DNase II gene could affect in vivo DNase II activity through reduction of the promoter activity, it is feasible to identify these SNPs susceptible to RA.

Serum deoxyribonuclease I activity can be a useful diagnostic marker for the early diagnosis of unstable angina pectoris or non–ST-segment elevation myocardial infarction

Recently, serum deoxyribonuclease I (DNase I) activity has been highlighted as a potential diagnostic marker for transient myocardial ischemia. To evaluate whether serum DNase I activity can be a useful biomarker for diagnosing unstable angina pectoris (UAP) or non-ST-segment elevation myocardial infarction (NSTEMI), we investigated serial changes in DNase I levels after chest pain in UAP and NSTEMI (UAP/NSTEMI) patients. Thirty-three and ten patients classified into the UAP/NSTEMI and the chest pain syndrome (CPS) group, respectively, were enrolled. The serum DNase I activity levels within 3h after chest pain and the absolute median value of percentage differences in serum DNase I activity levels from admission to 3h after hospitalization in the UAP/NSTEMI patients was significantly higher than those in the CPS patients. We evaluated the patients to show positive results for DNase I activity if the serum levels or percentage differences exceeded the corresponding cut-off values. The sensitivity and specificity of DNase I within 6h after chest pain in the UAP/NSTEMI patients without elevated levels of cardiac troponin T and the MB isoenzyme of creatine kinase were 89% and 88%, respectively. Serum DNase I activity can be a useful marker for the early diagnosis of UAP/NSTEMI after the onset of chest pain, irrespective of the evidence of myocardial injury.

Self-assembled cationic triblock copolymer mPEG-b-PDLLA-b-PDMA nanoparticles as nonviral gene vector

In this study, well-defined amphiphilic cationic triblock copolymers with different lengths of polycationic chain, methoxy poly(ethylene glycol)-b-poly(D,L-lactide)-b-poly(2-dimethylaminoethyl methacrylate) (mPEG-b-PDLLA-b-PDMA), were synthesized and evaluated as carriers for gene delivery. The prepared copolymers can self-assemble into spherical core–shell nanoparticles (NPs). The NPs have a very low critical micelle concentration (CMC) value with only 0.025 mg mL−1. Both copolymers can completely condense the pDNA into spherical complexes when the N/P ratio is equal to or above 3. Bovine serum albumin challenging and DNase I protection assay results demonstrate the mPEG-b-PDLLA-b-PDMA NPs can effectively protect the DNA against protein and nucleases. MTT assay results indicate that mPEG-b-PDLLA-b-PDMA NP/pDNA complexes exhibit obviously lower cytotoxicity compared with commercial gene transfection reagent Lipofectamine 2000/pDNA complexes. Subsequently, in vitro gene transfection studies in HeLa cells without serum show that mPEG113-b-PDLLA10-b-PDMA120 NP/pDNA complexes exhibit higher transfection efficiency than Lipofectamine 2000. Although the transfection efficiency is lower than that in the absence of serum, mPEG113-b-PDLLA10-b-PDMA120 NPs still display equivalent gene transfection efficiency compared to Lipofectamine 2000 when N/P ratio is above 15 in DMEM with 10% serum.

P.3.b.003 Decreased serum DNase I activity in patients with schizophrenia

P.3.b.003 Decreased serum DNase I activity in patients with schizophrenia

Neutrophil extracellular traps

Neutrophil extracellular traps

Functional and Genetic Survey of All Known Single-Nucleotide Polymorphisms Within the Human Deoxyribonuclease I Gene in Wide-Ranging Ethnic Groups

The single-nucleotide polymorphisms (SNPs) in the human DNase I gene (DNASE1) might be involved in susceptibility to some common diseases; however, only limited population data are available. Further, the effects of these SNPs on in vivo DNase I activity remain unknown. The genotype and haplotype of all the SNPs in DNASE1 were determined in 3 ethnic groups including 14 populations using newly developed methods. Together with our previous data on the nonsynonymous SNPs, two major haplotypes based on the five exonic SNPs were identified; genetic diversity in the Asian population was low. Among 10 SNPs, other than exonic SNPs in the gene, only 3 were polymorphic among all the populations. Haplotype distribution, based on all the polymorphic SNPs, was clarified to be generally varied in an ethnic-dependent manner. Thus, the genetic aspects of DNASE1 with regard to all the SNPs in wide-ranging ethnic groups could be first demonstrated. Further, there was no correlation of all the polymorphic SNPs other than nonsynonymous ones with serum DNase I activity levels. Polymorphic SNPs other than the exonic SNPs might not be directly related to common diseases through alterations in in vivo levels of the activity.

Impaired deoxyribonuclease I activity in patients with inflammatory bowel diseases.

Background and Aims. Deoxyribonuclease I (DNaseI) is an endonuclease that facilitates chromatin breakdown and promotes susceptibility to autoimmune disorders. The aim of current study was to investigate serum DNase I activity in patients with inflammatory bowel diseases (IBD). Patients and Methods. A cohort of 110 IBD patients was evaluated, aged 35 ± 12 years, 77 with Crohn's disease (CD) and 33 with ulcerative colitis (UC). 50 SLE patients and 50 healthy blood donors were examined as control groups. Results. DNase I activity in IBD patients was significantly lower than in healthy individuals, but higher than in SLE patients (P < .0001). Patients with UC showed higher DNase I activity than CD patients, P = .21. DNase I activity in female patients with IBD was significantly lower than in males, P = .024; however, no differences in DNase I activity were found in relation to gender in healthy individuals. DNase I activity has shown a strong negative correlation with the serum concentration of anti-nucleosomal antibodies in the autoimmune (SLE + IBD) cohort, as well as in the separate IBD cohort. Conclusions. Reduced serum DNase I activity probably has pathogenetic consequences in IBD. Induction of autoantibodies towards nucleosomes could be a reflection of impaired DNase I activity.

Development of a sensitive enzyme-linked immunosorbent assay for measurement of DNase I in human serum

Background Serum deoxyribonuclease I (DNase I) activity was reported to increase in the early phase after onset of acute myocardial infarction (AMI). Up to now, DNase I activity has been quantified by the single radial enzyme diffusion (SRED) method, which unfortunately requires a long incubation time. Therefore it is necessary to develop another assay suitable for measurement of serum DNase I concentrations in a clinical setting. Methods A sandwich ELISA was established for measurement of DNase I protein using a polyclonal antibody directed against DNase I protein and a biotinylated monoclonal for subsequent detection. Concentrations of serum DNase I protein were measured in healthy individuals and patients with AMI. Results This method was as precise as SRED, and took less time than SRED. A significant correlation was observed between DNase I concentration and enzyme activity ( r = 0.839; P < 0.001). The average of serum DNase I in AMI patients within 0–12 h of chest pain was significantly higher than that in healthy individuals ( P < 0.001), and decreased with time. Conclusions We have developed a sensitive ELISA capable of measuring DNase I protein concentrations. This method may be a useful alternative to SRED as an aid to diagnosis of AMI based on the serum DNase I level.

Serum deoxyribonuclease I can be used as a useful marker for diagnosis of death due to ischemic heart disease

We recently reported abrupt elevation of serum deoxyribonuclease I (DNase I) activity in the early stage of acute myocardial infarction (AMI). The aim of this study was to evaluate whether serum DNase I could be used as a marker for detection of myocardial ischemia that accompanies AMI. In consecutive patients with stable angina pectoris undergoing elective percutaneous coronary intervention (PCI) or the vasospastic angina pectoris (VSAP) provocation test, together with patients undergoing coronary angiography (CAG), serum samples were tested for DNase I activity. Serum DNase I activity was found to rise significantly within 3 h after PCI, and also after the provocation test in the VSAP-positive group. In all of the CAG and VSAP-negative patients, DNase I activity levels remained unchanged. Transient myocardial ischemia resulting from PCI or VSAP induces a significant elevation of serum DNase I activity, indicating that serum DNase I may be applicable as a novel and specific marker for detection of myocardial ischemia. Similarly, it is suggested that serum DNase I could be potentially useful as a biochemical marker for diagnosis of death due to ischemic heart disease.

Serum DNase I, soluble Fas/FasL levels and cell surface Fas expression in patients with SLE: a possible explanation for the lack of efficacy of hrDNase I treatment

The objectives of the study are to evaluate DNase I serum levels and their correlation with soluble Fas (sFas) and soluble Fas ligand (sFasL) and with cell surface Fas expression in patients with systemic lupus erythematosus (SLE), thus contributing to the dysregulated apoptosis typical of the disease. The methods include the following: Serum DNase I levels in patients and in controls were detected using the dot blot method and quantified by densitometry; sFas and sFasL were quantified using an ELISA system. Cell surface Fas expression was evaluated by FACS analysis. Apoptosis was studied by means of internucleosomal DNA degradation using a commercially available kit. The results are as follows: We found a significant difference in DNase I, sFas and sFasL serum levels between patients and controls. Levels of DNase I <7.79 ng ml(-1) are more represented in patients with SLE. Active SLE is strongly associated with high sFas levels and detectable sFasL. DNase I does not correlate with sFas or sFasL, whereas it correlates with T cell surface Fas expression that is higher in patients with active SLE than in healthy controls. Finally, administration of exogenous human recombinant DNase (hrDNase) I to freshly isolated T cells up-regulates cell surface Fas expression and induces increased susceptibility to Fas-mediated apoptosis. In conclusion, our findings confirm that DNase I is low in SLE and suggest that it may play a role in apoptosis in SLE by regulating the surface expression of the cell death molecule Fas. This role may contribute to explain the inefficacy of hrDNase I in SLE, a treatment proposed for the ability of DNase I to remove DNA from auto-antigenic nucleoprotein complexes.

Levels of serum deoxyribonuclease I activity on admission in patients with acute myocardial infarction can be useful in predicting left ventricular enlargement due to remodeling

Serum deoxyribonuclease I (DNase I) activity has recently been highlighted as a potential diagnostic marker for the early detection of an acute myocardial infarction (AMI). We evaluated whether the serum DNase I activity was associated with the parameters of the left ventricular (LV) remodeling after an AMI. We measured the serum DNase I activity in 45 patients with an AMI who were admitted to our hospital within approximately 4 h of the onset of their chest pain. We also evaluated the LV ejection fraction (LVEF), LV end-diastolic volume (LVEDV), and LV end-systolic volume (LVESV) of each patient by echocardiography at the time of admission and at 6 months after the onset of the AMI. The serum DNase I activity peaked at 3.5+/-2.0 h after the onset of the symptoms in the patients with an AMI, thereafter exhibiting a time-dependent decline within 12 h, and a return to the basal level within 24 h in almost all cases. Neither the LVEF, LVEDV, nor LVESV in each patient on admission exhibited a significant correlation to the peak levels of the serum DNase I activity. Although there was no correlation between the peak DNase I activity and LVEF at 6 months after the onset, a significant positive correlation of the peak DNase I activity with LVEDV and LVESV (r=0.48, p<0.001 and r=0.34, p=0.02, respectively) was found. Furthermore, the LVEDV at 6 months after the onset in the high DNase I activity group (> 17.9 U/L) were significantly higher than those in the low DNase I activity group (< or = 17.9 U/L) (118.0+/-28.2 ml vs 89.3+/-25.4 ml, p=0.026). The serum DNase I activity level may predict LV enlargement associated with remodeling after an AMI.

A novel mutation in the DNASE1 gene is related with protein instability and decreased enzyme activity in thyroid autoimmunity

A deficiency in the DNase enzyme, and thereby, a failure to remove DNA from nuclear antigens promotes disease susceptibility to autoimmune disorders. This study examined in patients with autoimmune thyroid disease (AITD) whether a reduced DNase activity is associated with sequence variations in the DNASE1 gene. The study included 18 patients with AITD, their 10 relatives, and 111 unrelated healthy controls. Serum DNase activity was determined with a validated, standardized enzyme-linked-immunosorbent assay. The promoter and all nine exons of the DNASE1 gene were sequenced. Heat stability of DNase enzyme was tested. In patients with AITD, a novel mutation (1218G > A, exon 5) and multiple polymorphisms were identified in the DNASE1 gene. The allele frequency of the mutation was increased in patients vs controls ( P = 0.001). In contrast to controls, the novel mutation was present in all five members of a family with AITD showing decreased DNase activity. The mutation resulted in the replacement of highly conserved valine with methionine at amino acid position 89 of the DNase enzyme. It was related to lowered heat stability and lowered activity of the enzyme. The identified new mutation and numerous polymorphisms, noted for the first time in AITD patients, may alter transcription and translation of the DNASE1 gene, thereby decreasing the stability and activity of the corresponding enzyme.

Characterization of PLL-g-PEG-DNA Nanoparticles for the Delivery of Therapeutic DNA

Local and controlled DNA release is a critical issue in current gene therapy. As viral gene delivery systems are associated with severe security problems, nonviral gene delivery vehicles were developed. Here, DNA-nanoparticles using grafted copolymers of PLL and PEG to increase their biocompatibility and stealth properties were systematically studied. Ten different PLL-based polymers with no, low, and high PEG grafting and PEG molecular weights as well as different PLL backbone lengths were complexed with plasmids containing 3200 to 10,100 base pairs. Stable complexes were formed and selected for cytotoxicity and transfection efficiency. Predominantly, PLL-g-PEG-DNA nanoparticles grafted with 4 or 5% PEG moieties of 5 kDa transfected 40% COS-7 cells without reduction of cell viability when formed at N/P ratios between 0.1 and 12.5. The molecular weight of PLL did not significantly affect transfection efficiency or cytotoxicity indicating that a specific cationic charge-density-to-PEG-ratio is important for efficient transfection and low cytotoxicity. The PLL-g-PEG-DNA nanoparticles were spherical with a diameter of approximately 100 nm and did not aggregate over 2 weeks. Moreover, they protected included plasmid DNA against serum components and DNase I digestion. Therefore, such storage stable and versatile PLL-g-PEG-DNA nanoparticles might be useful to deliver differently sized therapeutic DNA for in vivo applications.

Serum deoxyribonuclease I activity can be used as a novel marker of transient myocardial ischaemia: results in vasospastic angina pectoris induced by provocation test

Serum deoxyribonuclease I (DNase I) activity has recently been highlighted as a potential diagnostic marker for detection of acute myocardial infarction. To evaluate whether serum DNase I activity is useful for detection of myocardial ischaemia, we investigated alteration of its levels after onset of vasospastic angina pectoris (VSAP), resulting in transient myocardial ischaemia, induced by the intracoronary ergonovine provocation test. Twenty-nine consecutive patients with suspected VSAP were subjected to the test. Patients were categorized as VSAP-positive (n = 13) or -negative (n = 16) based on development of angina. Serum samples were examined for DNase I activity before, immediately after, and 3, 6, and 24 h after the provocation tests. The serum DNase I activity increased significantly from the baseline 3 h after the provocation test in 11 patients of the VSAP-positive group whose levels of troponin T were within the normal range. Median of the percentage differences from the baseline in serum DNase I activity 3 h after the test was 32.1% (25th and 75th percentile: 28.6 and 42.0%, respectively; P = 0.000012). In the VSAP-negative group, levels of DNase I activity remained unchanged at any point of time after the provocation test. Transient myocardial ischaemia resulting from VSAP induces a significant elevation of serum DNase I activity. Therefore, serum DNase I activity may be applicable as a useful marker for detecting transient myocardial ischaemia.