Cell-free mtDNA Research Articles

Diverse roles of mtDNA in schizophrenia: Implications in its pathophysiology and as biomarker for cognitive impairment

Schizophrenia (SZ) is a mental disorder characterized by neurocognitive dysfunctions and a reduction in occupational and social functioning. Several studies have provided evidence for mitochondrial dysfunction in the pathophysiology of SZ. In this sense, it is known that the addition of genetic variations in mitochondrial DNA (mtDNA) impairs oxidative phosphorylation of enzymatic complexes in mitochondria, resulting in ATP depletion and subsequent enhancement of reactive oxygen species; this is associated with cellular degeneration and apoptosis observed in some neuropsychiatric disorders. As a consequence of mitochondrial dysfunction, an increase in circulating cell-free mtDNA fragments can occur, which has been observed in individuals with SZ. Moreover, due to the bacterial origin of mitochondria, these cell-free mtDNA fragments in blood plasma may induce inflammatory and immunogenic responses, especially when their release is enhanced in specific disease conditions. However, the exact mechanism by which mtDNA could be released into blood plasma is not yet clear. Therefore, the aims of this review article were to discuss the participation of mtDNA genetic variations in physiopathologic mechanisms of SZ, and to determine the status of the disease and the possible ensuing changes over time by using circulating cell-free mtDNA fragments as a biomarker.

Mitochondrial DNA Stimulates TLR9-Dependent Neutrophil Extracellular Trap Formation in Primary Graft Dysfunction.

The immune system is designed to robustly respond to pathogenic stimuli but to be tolerant to endogenous ligands to not trigger autoimmunity. Here, we studied an endogenous damage-associated molecular pattern, mitochondrial DNA (mtDNA), during primary graft dysfunction (PGD) after lung transplantation. We hypothesized that cell-free mtDNA released during lung ischemia-reperfusion triggers neutrophil extracellular trap (NET) formation via TLR9 signaling. We found that mtDNA increases in the BAL fluid of experimental PGD (prolonged cold ischemia followed by orthotopic lung transplantation) and not in control transplants with minimal warm ischemia. The adoptive transfer of mtDNA into the minimal warm ischemia graft immediately before lung anastomosis induces NET formation and lung injury. TLR9 deficiency in neutrophils prevents mtDNA-induced NETs, and TLR9 deficiency in either the lung donor or recipient decreases NET formation and lung injury in the PGD model. Compared with human lung transplant recipients without PGD, severe PGD was associated with high levels of BAL mtDNA and NETs, with evidence of relative deficiency in DNaseI. We conclude that mtDNA released during lung ischemia-reperfusion triggers TLR9-dependent NET formation and drives lung injury. In PGD, DNaseI therapy has a potential dual benefit of neutralizing a major NET trigger (mtDNA) in addition to dismantling pathogenic NETs.

Clinical relevance of cell-free mitochondrial DNA during the early postoperative period in kidney transplant recipients

Recent studies indicate that urinary mitochondrial DNA (mtDNA) is predictive of ischemic AKI and is related to delayed graft function (DGF) in renal transplantation. Nevertheless, the clinical implications and prognostic value of urinary mtDNA in kidney transplantation remain undetermined. Here, we aimed to evaluate the associations between cell-free mtDNA and clinical parameters, including pathological findings in allograft biopsy and post-transplant renal function. A total of 85 renal transplant recipients were enrolled, and blood and urine samples were collected at a median of 17 days after transplantation. Cell-free nuclear and mtDNA levels were measured by quantitative polymerase chain reaction for LPL and ND1 genes. Urinary cell-free mtDNA levels were significantly higher in patients with DGF (P < 0.001) and cases of deceased donor transplantation (P < 0.001). The subjects with acute rejection showed higher urinary mtDNA levels than those without abnormalities (P = 0.043). In addition, allograft functions at 9- and 12-month post-transplantation were significantly different between tertile groups of mtDNA independent of the presence of DGF or acute rejection, showing significantly better graft outcome in the lowest tertile group. Urinary cell-free mtDNA levels during the early post-transplant period are significantly associated with DGF, acute rejection in graft biopsy, and short-term post-transplant renal function.

Plasma mitochondrial DNA is elevated in obese type 2 diabetes mellitus patients and correlates positively with insulin resistance.

Cells damaged by mechanical or infectious injury release proinflammatory mitochondrial DNA (mtDNA) fragments into the circulation. We evaluated the relation between plasma levels of mtDNA fragments in obese type 2 diabetes mellitus (T2DM) patients and measures of chronic inflammation and insulin resistance. In 10 obese T2DM patients and 12 healthy control (HC) subjects, we measured levels of plasma cell-free mtDNA with quantitative real-time polymerase chain reaction, and mtDNA damage in skeletal muscle with quantitative alkaline Southern blot. Also, markers of systemic inflammation and oxidative stress in skeletal muscle were measured. Plasma levels of mtDNA fragments, mtDNA damage in skeletal muscle and plasma tumor necrosis factor α levels were greater in obese T2DM patients than HC subjects. Also, the abundance of plasma mtDNA fragments in obese T2DM patients levels positively correlated with insulin resistance. To the best of our knowledge, this is the first published evidence that elevated level of plasma mtDNA fragments is associated with mtDNA damage and oxidative stress in skeletal muscle and correlates with insulin resistance in obese T2DM patients. Plasma mtDNA may be a useful biomarker for predicting and monitoring insulin resistance in obese patients.

Circulating Cell Free Nuclear DNA, Mitochondrial DNA and Global DNA Methylation: Potential Noninvasive Biomarkers for Breast Cancer Diagnosis

Eighty seven women with benign breast lesion, 120 patients with breast cancer (BC) and one hundred controls were included in the study. Quantification of mtDNA and nDNA was done by qPCR. Global DNA methylation was measured using ELISA. Circulating cell-free nDNA and mtDNA were significantly elevated in BC and benign breast lesions patients. Global methylation was significantly low in BC patients. Combining the studied parameters in one panel, nDNA/mtDNA/hypomethylation, improved their sensitivity in detecting BC to reach 92.5%. Circulating cell-free nDNA, mtDNA and global DNA hypomethylation can be used as diagnostic and prognostic markers for BC.

Simvastatin induces autophagic flux to restore cerulein-impaired phagosome-lysosome fusion in acute pancreatitis

BackgroundDuring pancreatitis, autophagy is activated, but lysosomal degradation of dysfunctional organelles including mitochondria is impaired, resulting in acinar cell death. Retrospective cohort analyses demonstrated an association between simvastatin use and decreased acute pancreatitis incidence. MethodsWe examined whether simvastatin can protect cell death induced by cerulein and the mechanisms involved during acute pancreatitis. Mice were pretreated with DMSO or simvastatin (20 mg/kg) for 24 h followed by 7 hourly cerulein injections and sacrificed 1 h after last injection to harvest blood and tissue for analysis. ResultsPancreatic histopathology revealed that simvastatin reduced necrotic cell death, inflammatory cell infiltration and edema. We found that cerulein triggered mitophagy with autophagosome formation in acinar cells. However, autophagosome-lysosome fusion was impaired due to altered levels of LAMP-1, AMPK and ULK-1, resulting in autophagosome accumulation (incomplete autophagy). Simvastatin abrogated these effects by upregulating LAMP-1 and activating AMPK which phosphorylated ULK-1, resulting in increased formation of functional autolysosomes. In contrast, autophagosomes accumulated in control group during pancreatitis. The effects of simvastatin to promote autophagic flux were inhibited by chloroquine. Mitochondria from simvastatin-treated mice were resistant to calcium overload compared to control, suggesting that simvastatin induced mitochondrial quality control to eliminate susceptible mitochondria. Clinical specimens showed a significant increase in cell-free mtDNA in plasma during pancreatitis compared to normal controls. Furthermore, genetic deletion of parkin abrogated the benefits of simvastatin. ConclusionOur findings reveal the novel role of simvastatin in enhancing autophagic flux to prevent pancreatic cell injury and pancreatitis.

Circulating Mitochondrial DNA as Predictor of Mortality in Critically Ill Patients: A Systematic Review of Clinical Studies

Despite numerous publications on mitochondrial DNA (mtDNA) in the last decade it remains to be seen whether mtDNA can be used clinically. We conducted a systematic review to assess circulating cell-free mtDNA as a biomarker of mortality in critically ill patients. This systematic review was registered with PROSPERO (CRD42016046670). PubMed, CINAHL, the Cochrane Library, Embase, Scopus, and Web of Science, and reference lists of retrieved articles were searched. Studies measuring circulating cell-free mtDNA and reporting on all-cause mortality in critically ill adult and pediatric patients were included. The primary and secondary outcomes were mortality and morbidity, respectively. Of the 1,566 initially retrieved publications, 40 studies were included, accounting for 3,450 critically ill patients. Substantial differences between studies were noted in how mtDNA was isolated and measured. Sixteen of the 40 included studies (40%) explored the association between mtDNA levels and mortality; of those 16 studies, 11 (68.8%) reported a statistically significant association. The area under the receiver operating characteristic (AUROC) curve for mtDNA and mortality was calculated for 10 studies and ranged from 0.61 to 0.95. There is growing interest in mtDNA as a predictor of mortality in critically ill patients. Most studies are small, lack validation cohorts, and utilize different protocols to measure mtDNA. When reported, AUROC analysis usually suggests a statistically significant association between mtDNA and mortality. Standardization of mtDNA protocols and the completion of a large, prospective, multicenter trial may be warranted to firmly establish the clinical usefulness of mtDNA.

Effects of resveratrol treatment on mitochondria and subsequent embryonic development of bovine blastocysts cryopreserved by slow freezing.

This study evaluated the effects of cryopreservation by slow freezing on the mitochondrial function, DNA integrity, and developmental ability of bovine embryos and examined whether resveratrol treatment of the frozen-thawed blastocysts improved embryonic viability. In vitro produced bovine embryos were subjected to slow freezing. After thawing, the ATP content and mitochondrial DNA integrity (mtDNA), determined by real-time PCR targeting short and long mitochondrial sequences, was found to be lower in frozen-thawed embryos than in fresh embryos, and mtDNA copy number was significantly reduced during the 24-hr incubation post warming. Furthermore, immunostaining against double-strand DNA revealed DNA damage in frozen-thawed embryos. When frozen-thawed embryos were incubated in the medium containing 0.5µM resveratrol, SIRT1 expression, and survival rate of the embryos significantly improved compared with the vehicle-treated embryos. In addition, cell-free mtDNA content in medium was higher in case of resveratrol-treated embryos than of vehicle-treated embryos. In conclusion, slow freezing affects mitochondrial integrity and function in the blastocysts. In the frozen-thawed embryos, mitochondria were removed during post-thawing incubation and resveratrol enhanced the process, resulting in improved survivability of the embryos.

Predictors of ccf-mtDNA reactivity to acute psychological stress identified using machine learning classifiers: A proof-of-concept

ObjectiveWe have previously found that acute psychological stress may affect mitochondria and trigger an increase in serum mitochondrial DNA, known as circulating cell-free mtDNA (ccf-mtDNA). Similar to other stress reactivity measures, there are substantial unexplained inter-individual differences in the magnitude of ccf-mtDNA reactivity, as well as within-person differences across different occasions of testing. Here, we sought to identify psychological and physiological predictors of ccf-mtDNA reactivity using machine learning-based multivariate classifiers. MethodWe used data from serum ccf-mtDNA concentration measured pre- and post-stress in 46 healthy midlife adults tested on two separate occasions. To identify variables predicting the magnitude of ccf-mtDNA reactivity, two multivariate classification models, partial least-squares discriminant analysis (PLS-DA) and random forest (RF), were trained to discriminate between high and low ccf-mtDNA responders. Potential predictors used in the models included state variables such as physiological measures and affective states, and trait variables such as sex and personality measures. Variables identified across both models were considered to be predictors of ccf-mtDNA reactivity and selected for downstream analyses. ResultsIdentified predictors were significantly enriched for state over trait measures (X2 = 7.03; p = 0.008) and for physiological over psychological measures (X2 = 4.36; p = 0.04). High responders were more likely to be male (X2 = 26.95; p < 0.001) and differed from low-responders on baseline cardiovascular and autonomic measures, and on stress-induced reduction in fatigue (Cohen's d = 0.38−0.73). These group-level findings also accurately accounted for within-person differences in 90% of cases. ConclusionThese results suggest that acute cardiovascular and psychological indices, rather than stable individual traits, predict stress-induced ccf-mtDNA reactivity. This work provides a proof-of-concept that machine learning approaches can be used to explore determinants of inter-individual and within-person differences in stress psychophysiology.

Detection of cell-free, exosomal and whole blood mitochondrial DNA copy number in plasma or whole blood of patients with serous epithelial ovarian cancer

Ovarian tumor is one of the leading causes of cancer among women. Patients are diagnosed at an advanced stage, usually. There is a need for new specific and sensitive biomarkers.Mitochondrial DNA copy number change was observed in various cancers. Our aim was to detect mitochondrial DNA copy number in whole blood (wb-mtDNA) and in plasma (cell-free and exosome encapsulated mtDNA) in patients with serous epithelial ovarian tumor.DNA was isolated from EDTA blood and plasma obtained from 24 patients and 24 healthy controls. Exosomes were isolated from cell-free plasma, and exosome encapsulated DNA (exoDNA) was extracted. Quantitative-real-time PCR was performed with Human Mitochondrial DNA (mtDNA) Monitoring Primer Set. Kruskall‑Wallis and Mann‑Whitney U test were used for data analysis.Wb-mtDNA copy number was significantly different among healthy controls and patients in multiple comparison (p = 0.0090 considering FIGO stage independently, and p = 0.0048 considering early- and late-stage cancers). There was a significant decrease among early-stage, all advanced stage and all cancer patients (FIGO I: 32.5 ± 8.3, p = 0.0061; FIGO III + IV: 37.2 ± 13.7 p = 0.0139; FIGO I + III + IV: 35.6 ± 12.2, p = 0.0017) or FIGO III patients alone (32.8 ± 5.6, p = 0.00089) compared to healthy controls.We found significant increase in copy number in exosomal mtDNA in cancer patients (236.0 ± 499.0, p = 0.0155), advanced-stage cancer patients (333.0 ± 575.0, p = 0.0095), of FIGO III (362.0 ± 609.2, p = 0.0494), and FIGO IV (304.0 ± 585.0, p = 0.0393) patients alone but not in samples of FIGO I patients (10.0 ± 3.5, p = 0.3907). In multiple comparison the increase was significant considering early- and late-stage cancers (p = 0.0253). Cell-free mtDNA copy numbers were not increased significantly.We found the highest copy number of mtDNA in exosomes, followed by plasma and peripheral blood in late-stage cancer patients. We observed significant difference in wb-mtDNA copy number between healthy controls and both early- and late-stage cancer patients.

Dialysate cell-free mitochondrial DNA fragments as a marker of intraperitoneal inflammation and peritoneal solute transport rate in peritoneal dialysis

BackgroundMitochondrial DNA (mtDNA) released into extracellular subsequent to cell injury and death can promote inflammation in patients and animal models. However, the effects of peritoneal dialysate cell-free mtDNA on intraperitoneal inflammation and peritoneal solute transport rate (PSTR) in peritoneal dialysis (PD) patients remain unclear.MethodsWe select the incident patients who began PD therapy between January 1, 2009, and December 30, 2010. Peritoneal dialysate was collected at the time of peritoneal equilibration test. The cell-free mtDNA, IL-6, IL-17A, TNF-α and IFN-γ were measured. All patients were followed till December 2017. The results were compared with PSTR and patient survival.ResultsOne hundred and eighty-nine patients were included in the study. The average age was 47.1 ± 13.5 years, 55.6% of the patients were males. The average PSTR was 0.66 ± 0.12, the median dialysate mtDNA levels were 4325 copies/ul. The median concentrations of IL-6, IL-17A, TNF-α and IFN-γ were 25.9, 10.8, 25.8 and 17.9 pg/ml, respectively. We found that dialysate mtDNA was significantly correlated with PSTR (r = 0.461, P < 0.001), IL-6 (r = 0.568, P < 0.001), TNF-α (r = 0.454, P < 0.001) and IFN-γ (r = 0.203, P = 0.005). After adjustment for multiple covariates, dialysate mtDNA levels were independently correlated with IL-6 and PSTR. Dialysate mtDNA levels were not associated with patient survival.ConclusionsWe found that dialysate mtDNA levels correlated with the degree of intraperitoneal inflammatory status in PD patients. Peritoneal effluent mtDNA was an independent determinant of PSTR but did not affect patient survival.

Circulating Cell-Free mtDNA Contributes to AIM2 Inflammasome-Mediated Chronic Inflammation in Patients with Type 2 Diabetes.

Mitochondrial dysfunction has been implicated in the pathogenesis of insulin resistance and type 2 diabetes. Damaged mitochondria DNA (mtDNA) may have a role in regulating hyperglycemia during type 2 diabetes. Circulating cell-free mitochondria DNA (ccf-mtDNA) was found in serum and plasma from patients and has been linked to the prognosis factors in various human diseases. However, the role of ccf-mtDNA in chronic inflammation in type 2 diabetes is unclear. In this study, we hypothesized that the ccf-mtDNA levels are associated with chronic inflammation in patients with type 2 diabetes. The mtDNA levels were elevated in the plasma from patients with type 2 diabetes compared to healthy subjects. The elevated mtDNA levels were associated with interleukin-1β (IL-1β) levels in patients with type 2 diabetes. The mtDNA, from patients with type 2 diabetes, induced absent in melanoma 2 (AIM2) inflammasome-dependent caspase-1 activation and IL-1β and IL-18 secretion in macrophages. Our results suggest that the ccf-mtDNA might contribute to AIM2 inflammasome-mediated chronic inflammation in type 2 diabetes.

Acute psychological stress increases serum circulating cell-free mitochondrial DNA

Intrinsic biological mechanisms transduce psychological stress into physiological adaptation that requires energy, but the role of mitochondria and mitochondrial DNA (mtDNA) in this process has not been defined in humans. Here, we show that similar to physical injury, exposure to psychological stress increases serum circulating cell-free mtDNA (ccf-mtDNA) levels. Healthy midlife adults exposed on two separate occasions to a brief psychological challenge exhibited a 2-3-fold increase in ccf-mtDNA, with no change in ccf-nuclear DNA levels, establishing the magnitude and specificity for ccf-mtDNA reactivity. In cell-based studies, we show that glucocorticoid signaling – a consequence of psychological stress in humans – is sufficient to induce mtDNA extrusion in a time frame consistent with stress-induced ccf-mtDNA increase. Collectively, these findings provide evidence that acute psychological stress induces ccf-mtDNA and implicate neuroendocrine signaling as a potential trigger for ccf-mtDNA release. Further controlled work is needed to confirm that observed increases in ccf-mtDNA result from stress exposure and to determine the functional significance of this effect.

Cell-Free Mitochondrial DNA in the CSF: A Potential Prognostic Biomarker of Anti-NMDAR Encephalitis.

Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is an autoimmune inflammatory brain disease that can develop a variety of neuropsychiatric presentations. However, the underlying nature of its inflammatory neuronal injury remains unclear. Mitochondrial DNA (mtDNA) is recently regarded as a damage-associated molecular pattern molecule (DAMP) that can initiate an inflammatory response. In the presenting study, we aimed to evaluate the levels of cell-free mtDNA in cerebrospinal fluid (CSF) of patients with anti-NMDAR encephalitis and to determine a potential role of cell-free mtDNA in the prognosis of anti-NMDAR encephalitis. A total of 33 patients with NMDAR encephalitis and 17 patients with other non-inflammatory disorders as controls were included in this study. The CSF levels of cell-free mtDNA were measured by quantitative polymerase chain reaction (qPCR). Cytokines including interleukin (IL)-6, IL-10, and tumor necrosis factor alpha (TNF-α) were measured by ELISA. The modified Rankin scale (mRS) score was evaluated for neurologic disabilities. Our data showed that the CSF levels of cell-free mtDNA and inflammation-associated cytokines were significantly higher in the patients with anti-NMDAR encephalitis compared with those in controls. Positive correlations were detected between the CSF levels of cell-free mtDNA and mRS scores of patients with anti-NMDAR encephalitis at both their admission and 6-month follow up. These findings suggest that the CSF level of cell-free mtDNA reflects the underlying neuroinflammatory process in patients with anti-NMDAR encephalitis and correlates with their clinical mRS scores. Therefore, cell-free mtDNA may be a potential prognostic biomarker for anti-NMDAR encephalitis.

Plasma mitochondrial DNA and metabolomic alterations in severe critical illness

BackgroundCell-free plasma mitochondrial DNA (mtDNA) levels are associated with endothelial dysfunction and differential outcomes in critical illness. A substantial alteration in metabolic homeostasis is commonly observed in severe critical illness. We hypothesized that metabolic profiles significantly differ between critically ill patients relative to their level of plasma mtDNA.MethodsWe performed a metabolomic study with biorepository plasma samples collected from 73 adults with systemic inflammatory response syndrome or sepsis at a single academic medical center. Patients were treated in a 20-bed medical ICU between 2008 and 2010. To identify key metabolites and metabolic pathways related to plasma NADH dehydrogenase 1 (ND1) mtDNA levels in critical illness, we first generated metabolomic data using gas and liquid chromatography-mass spectroscopy. We performed fold change analysis and volcano plot visualization based on false discovery rate-adjusted p values to evaluate the distribution of individual metabolite concentrations relative to ND1 mtDNA levels. We followed this by performing orthogonal partial least squares discriminant analysis to identify individual metabolites that discriminated ND1 mtDNA groups. We then interrogated the entire metabolomic profile using pathway overrepresentation analysis to identify groups of metabolite pathways that were different relative to ND1 mtDNA levels.ResultsMetabolomic profiles significantly differed in critically ill patients with ND1 mtDNA levels ≥ 3200 copies/μl plasma relative to those with an ND1 mtDNA level < 3200 copies/μl plasma. Several analytical strategies showed that patients with ND1 mtDNA levels ≥ 3200 copies/μl plasma had significant decreases in glycerophosphocholines and increases in short-chain acylcarnitines.ConclusionsDifferential metabolic profiles during critical illness are associated with cell-free plasma ND1 mtDNA levels that are indicative of cell damage. Elevated plasma ND1 mtDNA levels are associated with decreases in glycerophosphocholines and increases in short-chain acylcarnitines that reflect phospholipid metabolism dysregulation and decreased mitochondrial function, respectively.

Amniotic fluid cell-free DNA in preterm prelabor rupture of membranes.

We evaluated the levels of cell-free nuclear DNA (nDNA) and cell-free mitochondrial DNA (mtDNA) in the amniotic fluid supernatant from pregnancies complicated by preterm prelabor rupture of membranes (PPROM) based on evidence of microbial invasion of the amniotic cavity (MIAC) and/or intra-amniotic inflammation (IAI). A total of 155 women with PPROM were included in this study. Amniotic fluid samples were obtained by transabdominal amniocentesis. The levels of cell-free nDNA and mtDNA in the amniotic fluid supernatant were assessed and quantified by real-time polymerase chain reaction. The levels of cell-free nDNA and mtDNA were higher in women with MIAC and IAI than in women without these conditions (nDNA: with MIAC: median 3.9×104 genome equivalent [GE]/mL vs without MIAC: median 1.2×104 GE/mL, with IAI: median: 5.3×104 GE/mL vs without IAI: median 1.2×104 GE/mL; mtDNA: with MIAC: median 9.2×105 GE/mL vs without MIAC: median 2.5×105 GE/mL, with IAI: median 1.1×106 GE/mL vs without IAI: median 2.5×105 ; all P values≤0.01). Women with the microbial-associated IAI showed the highest levels of cell-free nDNA and mtDNA. Cell-free nDNA and mtDNA are constituents of the amniotic fluid supernatant from PPROM pregnancies. Both cell-free nDNA and mtDNA are involved in the intra-amniotic inflammatory response in women with PPROM.

Seminal plasma cell-free mitochondrial DNA copy number is associated with human semen quality

ObjectivePrevious studies have shown that mitochondrial DNA (mtDNA) copy number in human sperm is associated with semen quality. This study examines whether there is association between seminal cell-free mtDNA copy number and human semen parameters. Study designSeminal cell-free mtDNA copy number was measured in 205 men. Semen reactive oxygen species (ROS) level was also measured. Sperm quality was assessed using World Health Organization criteria, while sperm motility parameters were determined by computer-aided sperm analysis. ResultsComparing with normozoospermia, patients with asthenozoospermia and oligoasthenozoospermia had a significant decrease in cell-free mtDNA copy number and an increase in ROS level in seminal plasma (P < 0.05 for all). Seminal cell-free mtDNA copy number positively correlated with sperm concentration, motility, morphology and motion characteristics (r for 0.247 to 0.673, P < 0.05 for all). Semen ROS level negatively correlated with sperm concentration, motility, and motion characteristics (r for −0.261 to −0.676, P < 0.05 for all). There is a negative relationship between seminal cell-free mtDNA copy number and ROS level (r= -0.573, P < 0.05). ConclusionThe results suggested that seminal cell-free mtDNA copy number is associated with semen parameters, which may serve as a novel diagnostic marker of semen quality.

Cell free mitochondrial DNA in serum and milk associated with bovine mastitis: a pilot study

Mastitis is inflammation of mammary gland affecting all the species of domestic animals. Fragments of the mitochondrial genome released from dying cells are considered surrogate markers of mitochondrial injury. We hypothesized that bovine mastitis would be associated with increased cell free mitochondrial DNA (mtDNA) content in serum and milk. Milk and serum samples were collected from sub-clinical mastitic and normal animals. Mastitis was confirmed by California mastitis test and bacterial isolation. Oxidative stress, nitric oxide and inflammatory cytokines were also estimated. Real time polymerase chain reaction was conducted in serum and milk from sub-clinical mastitic animals and compared with healthy animals targeting the mtDNA genes cytochrome b. Mastitis animals showed higher oxidative stress markers and nitric oxide along with higher level of inflammatory cytokines. Cell free mtDNA was significantly higher in serum and milk of mastitic animals comparing to that of healthy control. The higher cell free relative mtDNA content in mastitis animals indicates injury to the mammary epithelial cells and thereby releasing the mtDNA in the milk and blood. This mtDNA may be a bio-marker of oxidative stress and tissue injury in bovine mastitis.

Reduced deoxyribonuclease enzyme activity in response to high postinjury mitochondrial DNA concentration provides a therapeutic target for Systemic Inflammatory Response Syndrome.

Cell-free mitochondrial DNA (mtDNA) is proinflammatory and has been detected in high concentrations in trauma patients' plasma. Deoxyribonuclease (DNAse) is the free plasma enzyme responsible for the digestion of extracellular DNA. The relationship between mtDNA and DNAse after major trauma is unknown. We hypothesized that DNAse activity would be elevated after injury and trauma surgery and would be associated with high concentrations of extracellular DNA. Two-year prospective study was performed on 103 consecutive trauma patients (male, 81%; age, 38 years [interquartile range, 30-59 years]; injury severity score, 18 [interquartile range, 12-26 years]) who underwent standardized major orthopedic trauma surgical interventions. Blood was collected at five perioperative time points (preoperative, postoperative, 7 hours, 24 hours, and 3 days postoperatively). Healthy control subjects (n = 20) were also sampled. Cell-free mtDNA and nuclear DNA (nDNA) were measured using quantitative polymerase chain reaction. Deoxyribonuclease was also assayed in the same plasma samples. Increased levels of mtDNA (from preoperative 163 ± 86 ng/mL to 3 days 282 ± 201 ng/mL, p < 0.0001) and nDNA (from preoperative 28 ± 20 ng/mL to 3 days 37 ± 27 ng/mL, p < 0.05) were present in trauma patients at all perioperative time points compared with healthy controls (mtDNA: 4 ± 2 ng/mL; nDNA: 10 ± 5 ng/mL). Deoxyribonuclease activity was lower in the trauma cohort (from preoperative 0.06 ± 0.04U/mL to 3 days 0.08 ± 0.04U/mL, p < 0.0001) compared with healthy controls (DNAse: 0.17 ± 0.03U/mL). There was no correlation between DNAse and perioperative DNA concentrations. Elevated mtDNA (but not nDNA) correlated with the development of systemic inflammatory response syndrome (SIRS) (p = 0.026) but not multiple organ failure. The significant perioperative elevation in plasma-free mtDNA concentration is associated with the development of SIRS. The fact that increased cell-free DNA concentrations present with significantly lower than healthy control DNAse activity suggests a potential therapeutic opportunity with DNAse administration to modulate postinjury severe SIRS. Prognostic/Epidemiological, level II.

Relationship between cardiovascular diseases and circulating cell-free nucleic acids in human plasma.

Cardiovascular diseases (CVDs) are the main cause of human morbidity and mortality worldwide. Early diagnosis could improve the efficiency of treatments. New biomarkers are needed for the identification of high-risk populations in order to make accurate diagnosis and therapy monitoring. Circulating cell-free nucleic acids (cf-NAs) offer a promising new noninvasive tool. These have a role in the regulation of normal physiological functions and in the development of pathological alterations. There is extended research on the clinical application and utilization of cell-free genomic DNA, mtDNA, mRNA, miRNA and long noncoding RNA in CVDs. These molecules could serve as components of new generation therapeutics. Our review focuses on the role of cf-NAs in the pathogenesis of CVDs and we are discussing also possible diagnostic applications and therapeutic implications.