Emotional Stress Induces Adaptive Response in Rat Lymphocytes to Subsequent Ionizing Radiation Exposure.

We studied the molecular mechanisms of cross-adaptation to ionizing radiation (1 Gy) of lymphocytes isolated from rats subjected to emotional stress. The effects of chronic (CES; various types of stress exposure) and acute (AES; forced swimming) emotional stress in rats on indicators of oxidative stress, cell death, and levels of NRF2 and NOX4 proteins involved in the development of the adaptive response were analyzed in isolated lymphocytes. It was found that stress induced an adaptive response in rat lymphocytes and triggered processes similar to the adaptive response induced by low doses of ionizing radiation: an increase in the level of oxidized DNA and cell death, as well as an increase in the content of NOX4 and NRF2 proteins. In animals subjected to emotional stress, suppressed DNA oxidation in response to irradiation, reduced levels of protective factor NRF2, as well as lymphocyte death were observed.

Copy number variations of satellite III (1q12) and ribosomal repeats in health and schizophrenia.

OBJECTIVE: Earlier we studied the copy number variations (CNVs) of ribosomal repeat (rDNA) and the satellite III fragment (1q12) (f-SatIII) in the cells of schizophrenia patients (SZ) and healthy controls (HC). In the present study we pursued two main objectives: (1) to confirm the increased rDNA and decreased f-SatIII content in the genomes of enlarged SZ and HC samples and (2) to compare the rDNA and f-SatIII content in the same DNA samples of SZ and HC individuals. METHODS: We determined the rDNA CN and f-SatIII content in the genomes of leukocytes of 1770 subjects [HC (N = 814) and SZ (N = 956)]. Non-radioactive quantitative hybridization method (NQH) was applied for analysis of the various combinations of the two repeats sizes in SZ and HC groups. RESULTS: f-SatIII in human leukocytes (N = 1556) varies between 5.7 and 44.7 pg/ng DNA. RDNA CN varies between 200 and 896 (N = 1770). SZ group significantly differ from the HC group by lower f-SatIII content and by rDNA abundance. The f-SatIII and rDNA CN are not randomly combined in the genome. Higher rDNA CN values are associated with higher f-SatIII index values in SZ and HC. The f-SatIII variation interval in SZ group increases significantly in the subgroup with the high rDNA CN index values (>300 copies). CONCLUSION: Schizophrenia patients' genomes contain low number of f-SatIII copies corresponding with a large ribosomal repeats CN. A scheme is proposed to explain the low f-SatIII content in SZ group against the background of high rDNA CN.

Accumulation of Circulating Cell-Free CpG-Enriched Ribosomal DNA Fragments on the Background of High Endonuclease Activity of Blood Plasma in Schizophrenic Patients.

INTRODUCTION: Schizophrenia (SZ) increases the level of cell death, leading to an increase in the concentration of circulating cell-free DNA (cfDNA). Ribosomal DNA (rDNA) contains many unmethylated CpG motifs that stimulate TLR9-MyD88-NF-κB signaling and the synthesis of proinflammatory cytokines. The number of rDNA copies in the genomes of SZ patients is increased; therefore, we expect that the concentration of cell-free rDNA in the plasma of the SZ patients also increases. This may be one of the explanations of the proinflammatory cytokine increase that is often observed in SZ. The major research question is what is the rDNA copy number in cfDNA (cf-rDNA CN) and its putative role in schizophrenia? Materials and Methods. We determined cfDNA concentration (RNase A/proteinase K/solvent extraction; fluorescent dye PicoGreen) and endonuclease activity (NA) of blood plasma (radial diffusion method) in the untreated male SZ group (N = 100) and in the male healthy control group (HC) (N = 96). Blood leukocyte DNA and cfDNA rDNA CN were determined with nonradioactive quantitative hybridization techniques. Plasma concentration of cf-rDNA was calculated. RESULTS: In the subjects from the SZ group, the mean cfDNA plasma concentration was twofold higher and NA of the plasma was fourfold higher than those in the healthy controls. rDNA CN in the blood leukocyte genome and in the cfDNA samples in the SZ group was significantly higher than that in the HC group. cf-rDNA concentration was threefold higher in the SZ group. CONCLUSION: Despite the abnormally high endonuclease activity in the blood plasma of SZ patients, the circulating cfDNA concentration is increased. Fragments of cf-rDNA accumulate in the blood plasma of SZ patients. Potentially, SZ patients' cfDNA should be a strong stimulating factor for the TLR9-MyD88-NF-κB signaling pathway.

Oxidized Cell-Free DNA Role in the Antioxidant Defense Mechanisms under Stress.

The present study focuses on the investigation of the oxidized cell-free DNA (cfDNA) properties in several experimental models, including cultured cerebellum cells, peripheral blood lymphocytes (PBL), plasma, and hippocampus under an acute and chronic unpredictable stress model in rats. Firstly, our study shows that Spectrum Green fluorescence-labeled oxidized cfDNA fragments were transferred into the cytoplasm of 80% of the cerebellum culture cells; meanwhile, the nonoxidized cfDNA fragments do not pass into the cells. Oxidized cfDNA stimulates the antioxidant mechanisms and induction of transcription factor NRF2 expression, followed by an activation of NRF2 signaling pathway genes-rise of Nrf2 and Hmox1 gene expression and consequently NRF2 protein synthesis. Secondly, we showed that stress increases plasma cfDNA concentration in rats corresponding with the duration of the stress exposure. At the same time, our study did not reveal any significant changes of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) level in PBL of rats under acute or chronic stress, probably due to the significantly increased Nrf2 expression, that we found in such conditions. 8-oxodG is one of the most reliable markers of DNA oxidation. We also found an increased level of 8-oxodG in the hippocampal homogenates and hippocampal dentate gyrus in rats subjected to acute and chronic stress. Taken together, our data shows that oxidized cfDNA may play a significant role in systemic and neuronal physiological mechanisms of stress and adaptation.

ROS-Induced DNA Damage Associates with Abundance of Mitochondrial DNA in White Blood Cells of the Untreated Schizophrenic Patients.

OBJECTIVE: The aim of this study was (1) to examine the leukocyte mtDNA copy number (CN) in unmedicated (SZ (m-)) and medicated (SZ (m+)) male patients with paranoid schizophrenia (SZ) in comparison with the healthy male controls (HC) and (2) to compare the leukocyte mtDNA CN with the content of an oxidation marker 8-oxodG in lymphocytes of the SZ (m-) patients. METHODS: We evaluated leukocyte mtDNA CN of 110 subjects with SZ in comparison with 60 male HC by the method qPCR (ratio mtDNA/nDNA (gene B2M) was detected). SZ patients were divided into two subgroups. The patients of the subgroups SZ (m+) (N = 55) were treated with standard antipsychotic medications in the hospital. The patients of the subgroup SZ (m-) (N = 55) were not treated before venous blood was sampled. To evaluate oxidative DNA damage, we quantified the levels of 8-oxodG in lymphocytes (flow cytometry) of SZ (m-) patients (N = 55) and HC (N = 30). RESULTS: The leukocyte mtDNA CN showed no significant difference in SZ (m+) patients and HC. The mtDNA CN in the unmedicated subgroup SZ (m-) was significantly higher than that in the SZ (m+) subgroup or in HC group. The level of 8-oxodG in the subgroup SZ (m-) was significantly higher than that in HC group. CONCLUSION: The leukocytes of the unmedicated SZ male patients with acute psychosis contain more mtDNA than the leukocytes of the male SZ patients treated with antipsychotic medications or the healthy controls. MtDNA content positively correlates with the level of 8-oxodG in the unmedicated SZ patients.

Abundance of ribosomal RNA gene copies in the genomes of schizophrenia patients.

OBJECTIVE: The ribosome is a critical component of the translation machinery. The key component of ribosomes is ribosomal RNA (rRNA). Dysregulation of rRNA biogenesis has been implicated in some human diseases. One of the factors affecting rRNA biogenesis is the ribosomal RNA genes (rDNA) copy number in the genome. The aim of this study was to examine the rDNA copy number (CN) variation in the genomes of patients with schizophrenia (SZ) compared to healthy controls (HC). METHODS: We evaluated rDNA CN in leukocytes of 179 subjects with SZ (108 male/71 female) in comparison with 122 HC (60 male/62 female) using two techniques: qPCR and nonradioactive quantitative hybridization (NQH), which is based on the use of biotinylated rDNA probes. RESULTS: rDNA CN (NQH) and rDNA CN (qPCR) was higher in SZ patients than in controls (median 542 vs 384, p=10-25 and median 498 vs 370, p=10-12). NQH was experimentally proved to be less sensitive to severe DNA damage than qPCR. The more DNA damage, the higher the ratio R=CN (NQH)/CN (qPCR). 15% of the SZ patients had significantly higher rDNA damage degree than the HC. CONCLUSION: Genomes of some SZ patients contain more ribosomal genes than those of HC. The elevated ribosomal genes copy number in human genome can be one of the genetic factors of schizophrenia development. This hypothesis requires further experimental studies to be corroborated or disproved.

Low-Dose Ionizing Radiation Affects Mesenchymal Stem Cells via Extracellular Oxidized Cell-Free DNA: A Possible Mediator of Bystander Effect and Adaptive Response.

We have hypothesized that the adaptive response to low doses of ionizing radiation (IR) is mediated by oxidized cell-free DNA (cfDNA) fragments. Here, we summarize our experimental evidence for this model. Studies involving measurements of ROS, expression of the NOX (superoxide radical production), induction of apoptosis and DNA double-strand breaks, antiapoptotic gene expression and cell cycle inhibition confirm this hypothesis. We have demonstrated that treatment of mesenchymal stem cells (MSCs) with low doses of IR (10 cGy) leads to cell death of part of cell population and release of oxidized cfDNA. cfDNA has the ability to penetrate into the cytoplasm of other cells. Oxidized cfDNA, like low doses of IR, induces oxidative stress, ROS production, ROS-induced oxidative modifications of nuclear DNA, DNA breaks, arrest of the cell cycle, activation of DNA reparation and antioxidant response, and inhibition of apoptosis. The MSCs pretreated with low dose of irradiation or oxidized cfDNA were equally effective in induction of adaptive response to challenge further dose of radiation. Our studies suggest that oxidized cfDNA is a signaling molecule in the stress signaling that mediates radiation-induced bystander effects and that it is an important component of the development of radioadaptive responses to low doses of IR.

Quantification of cell-free DNA in blood plasma and DNA damage degree in lymphocytes to evaluate dysregulation of apoptosis in schizophrenia patients.

Oxidative DNA damage has been proposed as one of the causes of schizophrenia (SZ), and post mortem data indicate a dysregulation of apoptosis in SZ patients. To evaluate apoptosis in vivo we quantified the concentration of plasma cell-free DNA (cfDNA index, determined using fluorescence), the levels of 8-oxodG in cfDNA (immunoassay) and lymphocytes (FL1-8-oxodG index, flow cytometry) of male patients with acute psychotic disorders: paranoid SZ (total N = 58), schizophreniform (N = 11) and alcohol-induced (N = 14) psychotic disorder, and 30 healthy males. CfDNA in SZ (N = 58) does not change compared with controls. In SZ patients. Elevated levels of 8-oxodG were found in cfDNA (N = 58) and lymphocytes (n = 45). The main sources of cfDNA are dying cells with oxidized DNA. Thus, the cfDNA/FL1-8-oxodG ratio shows the level of apoptosis in damaged cells. Two subgroups were identified among the SZ patients (n = 45). For SZ-1 (31%) and SZ-2 (69%) median values of cfDNA/FL1-8-oxodG index are related as 1:6 (p < 0.0000001). For the patients with other psychotic disorders and healthy controls, cfDNA/FL1-8-oxodG values were within the range of the values in SZ-2. Thus, apoptosis is impaired in approximately one-third of SZ patients. This leads to an increase in the number of cells with damaged DNA in the patient's body tissues and may be a contributing cause of acute psychotic disorder.

Functionalized Fullerene Increases NF-κB Activity and Blocks Genotoxic Effect of Oxidative Stress in Serum-Starving Human Embryo Lung Diploid Fibroblasts.

The influence of a water-soluble [60] fullerene derivative containing five residues of 3-phenylpropionic acid and a chlorine addend appended to the carbon cage (F-828) on serum-starving human embryo lung diploid fibroblasts (HELFs) was studied. Serum deprivation evokes oxidative stress in HELFs. Cultivation of serum-starving HELFs in the presence of 0.1-1 µM F-828 significantly decreases the level of free radicals, inhibits autophagy, and represses expression of NOX4 and NRF2 proteins. The activity of NF-κB substantially grows up in contrast to the suppressed NRF2 activity. In the presence of 0.2-0.25 µM F-828, the DSB rate and apoptosis level dramatically decrease. The maximum increase of proliferative activity of the HELFs and maximum activity of NF-κB are observed at these concentration values. Conclusion. Under the conditions of oxidative stress evoked by serum deprivation the water-soluble fullerene derivative F-828 used in concentrations of 0.1 to 1 µM strongly stimulates the NF-κB activity and represses the NRF2 activity in HELFs.

[The maternal effect in infantile autism: elevated DNA damage degree in patients and their mothers]. / Materinskii éffekt pri detskom autizme: povyshennyi uroven' povrezhdenii DNK u patsientov i ikh materei.

Infantile autism is a common disorder of mental development, which is characterized by impairments in the communicative, cognitive and speech spheres and obsessional stereotyped behaviour. Although in most cases, pathogenic factors remain unclear, infantile autism has a significant hereditary component, however, its etiology is also under the influence of environmental factors, including the condition of the mother's body during pregnancy ("maternal effect"). Oxidative stress is assumed to play a key role in the pathogenesis of infantile autism. It is known that oxidative stress has a prominent genotoxic effect, which is realized through inducing single and double strand breaks of the nuclear DNA. We evaluated the degree of DNA damage in patients with infantile autism and their mothers using DNA comet assay. The comet tail moment and DNA per cent ratio in the tail were assessed for each individual. The two parameters appeared to be strongly correlated (r=0.90). Mean and median values of both parameters were considerably higher in the sample of autistic children, than in age-matching healthy controls. Interestingly, these parameters were also elevated in healthy mothers of autistic children, with no difference from the values in the group of autistic children. The control group of healthy women of reproductive age, who had no children with autism, differed by the DNA comet tail moment from the group of mothers of autistic children, but did not differ significantly from the control group of healthy children. The results suggest that there are genotoxic factors in mentally healthy mothers of autistic children, which can determine the pathological process in the foeti via environmental "maternal effect" during gestation.

[Enrichment of extracellular DNA from the cultivation medium of human peripheral blood mononuclears with genomic CpG rich fragments results in increased cell production of IL-6 and TNF-a via activation of the NF-kB signaling pathway]. / Obogashchenie vnekletochnoi DNK sredy kul'tivirovaniia mononuklearov perifericheskoi krovi cheloveka CpG-bogatymi fragmentami genoma privodit k uvelicheniiu produktsii kletkami IL-6 i TNF-a putem aktivatsii signal'nogo puti NF-kB.

Previously, it was found that blood plasma extracellular DNA (ecDNA) of patients with rheumatoid arthritis (RA) is enriched with CpG-rich genomic DNA fragments, which contain TLR9 ligands (Veiko et al., 2006). In this study we have demonstrated that ecDNA of a RA patient and model fragments added to a cultivation medium of peripheral blood mononuclear cells (PBMC) of healthy donors stimulate expression of genes for the TLR9-MyD88-NF-kB signaling pathway; this leads to a significant increase in concentrations of the proinflammatory cytokines IL-6 and TNF-a in the cultivation medium. Human genomic DNA non-enriched with the CpG sequences did not stimulate IL-6 and TNF-a synthesis in PBMC. A scheme explaining the potential role ecDNA in the induction and maintenance of increased levels of the proinflammatory cytokines under conditions damaging the human cells has been proposed.

Toxic and DNA damaging effects of a functionalized fullerene in human embryonic lung fibroblasts.

Water-soluble fullerenes have been studied as potential nanovectors and therapeutic agents, but their possible toxicity is of concern. We have studied the effects of F-828, a soluble fullerene [C60] derivative, on diploid human embryonic lung fibroblasts (HELFs) in vitro. F-828 causes complex time-dependent changes in ROS levels. Inhibition of Nox4 activity by plumbagin blocks F-828-dependent ROS elevation. F-828 induces DNA breaks, as measured by the comet assay and γH2AX expression, and the activities of the transcription factors NF-kB and p53 increase. F-828 concentrations>25µM are cytotoxic; cell death occurs by necrosis. Expression levels of TGF-ß, RHOA, RHOC, ROCK1, and SMAD2 increase following exposure to F-828. Our results raise the possibility that fullerene F-828 may induce pulmonary fibrosis in vivo.

RSH ANTIOXIDANTS INCREASE INTRACELLULAR ROS LEVEL AND PROLIFERATION OF SP2/0 MYELOMA CELLS IN SERUM-FREE MEDIUM.

At present research we have demonstrated the link between introducing of low molecular weight RSH-antioxidants (N-acetylcysteine, glutathione) into serum-free medium composition, reactive oxygen species (ROS) generation and mouse myeloma SP2/0-SF cells proliferative activity. The presence of these compounds in the medium changed the pattern of ROS-activity in cells by concentration-dependent manner, and affected their proliferative characteristics. The optimal value of the proliferative activity was related to 0.2 mM for both thyols and not depended from the thyol-compound nature. Further increasing up from the found concentration optimum lead to growth inhibition with different expression for N-acetylcysteine and glutathione.

[Circulating in blood plasma cell-free DNA in the pathogenesis of ischemic stroke: the role of the transcribed region of ribosomal repeat].

It has been established that DNA, in addition to its basic functions (storage and realization of genetic information), also carries CpG-rich sequences having immunopotentiating properties. In this study we investigated the dynamics of the quantitative and qualitative characteristics of cell-free DNA (cfDNA) circulating in blood plasma of patients with acute ischemic stroke compared with the biomechanics of their blood samples flow, cerebral infarct volume and dynamics of neurological disorders. The results obtained revealed a new drag-reducing function of the circulating cfDNA and its important role in a regulation of blood flow hydrodynamic resistance in conditions of disturbed cerebral circulation. Moreover, our results showed a dependence of cerebral infarct volume and clinical picture dynamics on the plasma concentration of transcribed region of ribosomal repeat CpG-rich sequences (rDNA). It was established a new function of rDNA fragments circulating in the total pool cfDNK, i.e., generation of the intercommunication between blood and brain cells to induce neuroprotection in ischemic stroke.

Oxidized extracellular DNA suppresses nitric oxide production by endothelial NO synthase (eNOS) in human endothelial cells (HUVEC).

Circulating DNA from patients with cardiovascular diseases reduce the synthesis of NO in endothelial cells, which is probably related to oxidative modification of DNA. To test this hypothesis, HUVEC cells were cultured in the presence of DNA containing ~1 (nonoxidized DNA), 700, or 2100 8-oxodG/10(6) nucleosides. Nonoxidized DNA stimulated the synthesis of NO, which was associated with an increase in the expression of endothelial NO synthase. Oxidized NO decreased the amount of mRNA and protein for endothelial NO synthase, but increased the relative content of its low active form. These changes were accompanied by reduction of NO production. These findings suggest that oxidative modification of circulating extracellular DNA contributes to endothelial dysfunction manifested in suppression of NO production.

Properties of extracellular DNA from the cerebrospinal fluid and blood plasma during Parkinson's disease.

The cerebrospinal fluid of patients with Parkinson's disease was shown to contain extracellular DNA. Extracellular DNA concentration in the cerebrospinal fluid was 3.3-fold lower than in blood plasma from these patients. HPLC-mass spectrometry analysis showed that the pool of extracellular DNA from the liquor is characterized by a lower content of deoxythymidine, but greater amounts of deoxycytidine and deoxyguanosine than the pool of extracellular DNA from the plasma. The level of deoxyguanosine was 2 times lower than that of deoxycytidine (as differentiated from plasma extracellular DNA with similar content of these substances). Our findings indicate that extracellular DNA from the cerebrospinal fluid contains considerable amounts of modified deoxyguanosine. These data attest to significant differences in the quantitative and qualitative characteristics of extracellular DNA from the blood and cerebrospinal fluid of patients. Specific features of extracellular DNA from the cerebrospinal fluid of patients suggest its involvement in the pathogenesis of Parkinson's disease.

[Dimeric bisberzimidazoles: cytotoxicity and effects on DNA methylation in normal and cancer human cells].

Cancer cells are characterized by the hypermethylation of promoter regions of tumor suppressor genes. DNA methyltransferase inhibitors cause re-activation of these genes that allows considering DNA methyltransferases as targets for anticancer therapy. As it was previously shown by us, dimeric bisbenzimidazoles, DB(n), differing in length of the oligomethylene linker between the two bisbenzimidazole fragments (n--number of methylene groups in linker) effectively inhibit the methylation of DNA duplexes by murine methyltransferase Dnmt3a. Here, the cytotoxicity of some of these compounds, their penetration into cells and influence on the methylation of genomic DNA in fetal lung fibroblasts line F-977 and cervical cancer cells HeLa have been studied. In the 0-60 microM concentration range, only the DB(11) displayed a significant toxic effect on the normal cells, whereas the effect of DB(n) investigated on the cancer cells was not significant. Interestingly, the DB(1) and DB(3) to a small extent stimulate the proliferation of HeLa and F-977 cells, respectively. DB(1) and DB(3) display ability to penetrate into the nucleus of HeLa and F-977 cells and accumulate in various parts of the nuclei. DB(11) is not able to penetrate into the nuclei of these cells. The incubation of F-977 cells with 26 microM of DB(1) or DB(3) led to a decrease of the methylation of 18S rRNA gene, which is located in the region of DB(1) and DB(3) accumulation. A similar effect produces the same concentration of DB (3) in the F-977 cells. However, the overall level of genomic DNA methylation was not changed. These data suggest that DB(n) can be directed to act on specific genes demethylation and in the future may selectively inhibit the proliferation of cancer cells.

Emotional stress in rats changes concentration and composition of extracellular DNA circulating in blood plasma under normal conditions and in cerebral ischemia.

We studied quantitative and qualitative characteristics of extracellular DNA circulating in the blood plasma of Wistar rats under normal conditions, in psychoemotional stress (after 18 hours of aggressive conflict situation), and in acute cerebral ischemia. It was found that animals predisposed to psychoemotional stress normally have increased levels of antibodies against low excreted fragment of transcribed region of ribosomal DNA repeat rich in cytosine-guanine (CpG). A sharp increase in the level of circulating extracellular DNA was noted. Its increase was more pronounced during ischemia against the background of psychoemotional stress than in the control. These data suggest that multiple stress exposures experienced during the life can result in accumulation of GpG-rich sequences in the plasma of individuals predisposed to psychoemotional stress.

[Use of the most recent reagent (CuFL) for stimulation of NO synthesis by the medicinal leech salivary cell secretion in the cultures of human endothelium cells (HUVEC) and in rat cardiomiocytes].

The medicinal leech salivary cell secretion (SCS) may stimulate NO-production in cultures of human endothelium cells (HUVEC) and rat cardiomiocytes (RCM). This effect was detected using a NO specific reagent, - the complex Cu2+ with a fluorescein derivative (Cu-Fl). NO had also been detected in the cells by fluorescent electronic microscopy and determined quantitatively in the cells and in culture fluid by the fluorescence method. SCS stimulated NO synthesis in HUVEC cells (but not in RCM) is accompanied by NO release into intercellular space. Localization of NO synthesis centers is presented and it is shown that the increase in NO levels during the SCS action on HUVEC and RCM is associated with the increase in the activity of eNOS/nNOS, but not iNOS. In endothelial cells SCS activates nitrosylation processes, assessed by the increase of nitrite-ions in the culture medium. It is therefore important to use Cu-Fl, other than Griss-reagent, during the first hour of analysis of NO synthesis. The NO-depended mechanism of SCS action on endothelial cells might be a factor in providing of its positive action in hirudotheraphy.

[Cell-free DNA fragments increase transcription in human mesenchymal stem cells, activate TLR-dependent signal pathway and supress apoptosis].

Human mesenchymal stem cells (MSCs) are now widely adopted in regenerative medicine. However, many questions on the role of different signaling pathways in the regulation of stem cell (SC) functional activity within the organism remain unaswered. In damaged regions the level of cell death increases and DNA fragments from dead cells (cell-free DNA, cfDNA) are accumulated in blood. We showed that in adipose-derived MSCs exposed in vitro to cfDNA fragments the transcription level increased (the total amount of cellular RNA and the rRNA amount rose). GC-rich CfDNA fragments (GC-DNA) activated the TLR9-dependent signal pathway: the expression of TLR9 and of TLR9-signaling pathway adapter--MyD88--was up-regulated. AT-rich DNA fragments did not increase the TLR9 expression, though, the MyD88 expression level rose. So we suggest that AT-DNA acts via some other receptors that nevertheless activate MyD88-dependent signalling in MSCs. We also showed that cfDNA fragments decreased the activity of caspase, an apoptotic enzyme. So, ctDNA can significantly influence the functional activity ofMSC by activating TLR9- and MyD88-dependent signal pathways and lowering the apoptosis level.