Performance of a targeted cell-free DNA prenatal test for 22q11.2 deletion in a large clinical cohort.

OBJECTIVE: 22q11.2 deletion is more common than trisomies 18 and 13 combined, yet no routine approach to prenatal screening for this microdeletion has been established. This study evaluated the clinical sensitivity and specificity of a targeted cell-free DNA (cfDNA) test to screen for fetal 22q11.2 deletion in a large cohort, using blinded analysis of prospectively enrolled pregnancies and stored clinical samples. METHODS: In order to ensure that the analysis included a meaningful number of cases with fetal 22q11.2 deletion, maternal plasma samples were obtained by prospective, multicenter enrolment of pregnancies with a fetal cardiac abnormality and from stored clinical samples from a research sample bank. Fetal genetic status, as evaluated by microarray analysis, karyotyping with fluorescence in-situ hybridization or a comparable test, was available for all cases. Samples were processed as described previously for the Harmony prenatal test, with the addition of DANSR (Digital Analysis of Selected Regions) assays targeting the 3.0-Mb region of 22q11.2 associated with 22q11.2 deletion syndrome. Operators were blinded to fetal genetic status. Sensitivity and specificity of the cfDNA test for 22q11.2 deletion were calculated based on concordance between the cfDNA result and fetal genotype. RESULTS: The final study group consisted of 735 clinical samples, including 358 from prospectively enrolled pregnancies and 377 stored clinical samples. Of 46 maternal plasma samples from pregnancies with a 22q11.2 deletion, ranging in size from 1.25 to 3.25 Mb, 32 had a cfDNA result indicating a high probability of 22q11.2 deletion (sensitivity, 69.6% (95% CI, 55.2-80.9%)). All 689 maternal plasma samples without a 22q11.2 deletion were classified correctly by the cfDNA test as having no evidence of a 22q11.2 deletion (specificity, 100% (95% CI, 99.5-100%)). CONCLUSIONS: The results of this large-scale prospective clinical evaluation of the sensitivity and specificity of a targeted cfDNA test for fetal 22q11.2 deletion demonstrate that this test can detect the common and smaller, nested 22q11.2 deletions with a low (0-0.5%) false-positive rate. Although the positive predictive value (PPV) observed in this study population was 100%, the expected PPV in the general pregnant population is estimated to be 12.2% at 99.5% specificity and 41.1% at 99.9% specificity. The use of this cfDNA test to screen for 22q11.2 deletion could enhance identification of pregnancies at risk for 22q11.2 deletion syndrome without significantly increasing the likelihood of maternal anxiety and unnecessary invasive procedures related to a false-positive result. © 2021 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

[N-stearoylethanolamine effect on the level of 11-hydroxycorticosteroids, cytokines IL-1, IL-6 and TNFalpha in rats with nonspecific inflammation caused by thermal burn of skin].

The mechanisms of anti-inflammatory action of saturated N-acylethanolamine--N-stearoylethanolamine (NSE) were investigated on the rat model of nonspecific inflammation (thermal burns of the skin). The results showed that the NSE application in a form of aqueous suspension (10 mg/ml) on the damaged skin area during 12 days significantly accelerated the healing process of burned wounds. NSE also prevented the increase of 11-hydroxycorticosteroids content in the blood of rats with burns. There was also found a significant decrease of cytokines (IL-1beta, IL-6 and TNFalpha) levels under the NSE action. This way may be one of the mechanisms of NSE anti-inflammatory action.

[The effect of N-stearoylethanolamine on liver phospholipid composition of rats with insulin resistance caused by alimentary obesity].

We used alimentary obesity-induced insulin resistance (IR) model in rats to investigate the influence of N-stearoylethanolamine on the content of phospholipids and their fatty acid composition. Our results show that prolonged high-fat diet triggers considerable aberrations in the composition of main phospholipids in the liver and can be one of the causes of IR in rats. In particular, the increase of phosphatidylcholine, phosphatidylethanolamine and significant decrease of other phospholipids: lysophosphatidylcholine, lysophosphatidylethanolamine, sphingomyelin, phosphatidylinositol, phosphatidylserine and diphosphaglicerol were observed. The levels of monounsaturated (erucic, nervonic, oleic) and polyunsaturated (eicosatrienoic, docosatrienoic, arachidonic) fatty acids were increased; meanwhile the content of diunsaturated acids was decreased. The NSE administration (50 mg/kg of body weight) caused restoration of the phospholipids content in the liver of rats with diet-induced IR that highly correlated with the decrease in plasma insulin level and the improvement of insulin sensitivity. Moreover, the effect of NSE was accompanied by the normalization of fatty acids composition of phospholipids that could be related to modulating influence of NSE on the activity of the main fatty acid desaturases. It is known that the imbalance in phospholipid composition of the rat liver causes substantial metabolic alterations that are associated with the development of IR. Accordingly, the compensations of the imbalance by NSE can help to restore insulin sensitivity, inhibit the development of obesity, IR and type 2 diabetes.

[Effects of N-stearoylethanolamine on the emotionality and learning ability of rats].

After administration of endocannabinoid-like compound N-stearoylethanolamine (NSE) at a dose of 0,1 and 5 mg/kg the anxiety level of rats in the elevated plus maze and learning ability of rats in the radial maze were investigated. It was revealed that NSE can change both innate and acquired behavior of rats. It was found that the administration of NSE decreased of anxious behavior in general, although number and duration of grooming were not affected. Administration of NSE at a dose of 5 mg/kg decreases of anxious behavior in rats but also decreases locomotor activity. Higher anxiolytic effect of the substance in the elevated plus maze and growth of learning ability in the radial maze were shown during 7 days' introduction of NSE at a dose of 0,1 mg/kg that administration of this substance at a dose of 5 mg/kg. Administration of NSE at a dose of 0.1 mg/kg significantly reduced the total number of errors in the radial maze compared to the control at the first day of conditioning with food reinforcement. The latent period of 3rd reinforcement's taking in animals in this group was lower on the 1st, 5th (P<0.05) and 6th (P <0.01) days. Nevertheless, it was found no significant differences in the behavior in rats treated with NSE in both doses throughout the study period. Therefore, NSE changes the behavior of rats and contributes to the improvement of cognitive function without negative effects specific to cannabinoid drugs.

[Organ-specific antitoxic effects of N-stearoylethanolamine in male mice with Lewis carcinoma under doxorubicin intoxication].

With the introduction of doxorubicin into mice with Lewis carcinoma in the heart and liver tissues and kidney the organ-antitoxic effects of N-stearoilethanolamine (NSE) were found, which depended on its concentration. Administration of doxorubicin to male mice leads to an increase in the level of urea and creatinine, as well as activation of ALT in blood plasma. Introduction of NSE resulted in normalization of these parameters to the level of intact animals. In the heart tissue doxorubicin has multidirectional effects on the activity of antioxidant enzymes, in particular it decreases the activity of catalase and superoxide dismutase activity increases. Introduction of NSE normalizes these two indicators. It was found that tumor growth leads to an increase in the activity of glutathione peroxidase and superoxide dismutase. Introduction of NSE normalizes activity of these enzymes. Doxorubicin causes an increase in catalase activity in the kidney of mice with tumour, NSE prevented the increase in the activity of the above enzyme. The cancer process leads to increased levels of catalase activity in the liver of tumour-bearing mice, the introduction of NSE decreases the enzyme activity.

[The effect of N-stearoylethanolamine on the activity of antioxidant enzymes, content of lipid peroxidation products and nitric oxide in the blood plasma and liver of rats with induced insulin-resistance].

The influence of N-stearoylethanolamine (NSE) on the content of lipid peroxidation products, activity of antioxidant enzymes and the nitric oxide level in the liver and blood plasma of rats with insulin-resistance (IR) state was investigated. IR state was induced in rats by prolonged high-fat diet (58% of energy derived from fat) for 6 months combined with one injection of streptozotocin (15 mg/kg of body weight). The existence of IR state was estimated by results of glucoso-tolerance test and blood plasma insulin content. The level of lipid peroxides products was shown to be higher in the liver of insulin resistant animals as a result of reduced superoxide dismutase and catalase activity, however, glutathione peroxidase activity was increased. The increase of nitric-oxide content in the liver and blood plasma of high-fat diet rats compared with healthy control animals was also observed. The administration of the NSE suspension per os in a dose of 50 mg/kg during 2 weeks to the rats with induced insulin-resistance state contributed to the increase of superoxide dismutase, catalase and glutathione peroxidase activity. In consequence of antioxidant enzymes activation the intensity of POL process was decreased. The NSE administration caused normalization of nitric oxide level, restoring pro-/antioxidant balance in the liver and blood plasma of rats with IR state. In conclusion, the NSE administration to the rats with insulin-resistance state restored pro-/antioxidant balance and enhanced the content of nitric oxide, therefore, improving insulin sensitivity.

[Antitoxic and antioxidant effects of N-stearoylethanolamin in the content of nanocomposite complex with doxorubicin in organs of mice with Lewis carcinoma].

The aim of the study was to evaluate the possibility to reduce the doxorubicin toxic effects by its immobilization with N-stearoylethanolamine (NSE) on nanocarier polyethylene glycol. The studied parameters of the doxorubicin toxicity were: the level of creatinine in the mice blood plasma and activity of alanine aminotransferase and aspartate aminotransferase in the blood plasma of mice. The activity of catalase superoxide dismutase, glutathione peroxidase and intensity of lipid peroxidation was determined in the tissues of the heart, kidneys and liver. Doxorubicin in the content of nanocarrier alone caused an increase of serum creatinine and aspartateaminotrasferase activity in plasma of experimental animals with carcinoma. Nanocomposite which contained doxorubicin and NSE, did not cause an increase of these parameters. It has been shown that the administration of a carrier containing doxorubicin to mice with Lewis lung carcinoma caused the decrease of catalase activity in mice with carcinoma. The combination of NSE and doxorubicin on the carrier led to the normalization of this parameter to the level of intact animals. NSE immobilized on a carrier together with doxorubicin caused a decrease in the activity of superoxide dismutase in the kidney tissue of mice with tumor. The tumor growth caused the increase of the of superoxide dismutase in mice. The administration of a carrier which contained doxorubicin and NSE normalized superoxide dismutase in heart tissue contrary of kidney. The obtained results show the antitoxic and antioxidant effects of N-stearoylethanolamine immobilized in the nanocarrier complex together with doxorubicin.

[Protective effect of N-stearoylethanolamine in suspension and in nanocomposite complex in the organs of mice with the Lewis carcinoma under doxorubicin intoxication administration].

The antioxidant effects of N-stearoylethanolamine (NSE) in the nanocomplex composition and in suspension are shown on the model of intoxication by doxorubicin in conditions of development of the Lewis carcinoma in the heart, kidneys and liver tissue and in the blood plasma of female mice. The NSE suspension reduces the level of urea in the blood plasma of mice with the Lewis carcinoma, which growth was revealed as a result of introduction of doxorubicin. Under introduction of nanocomplex the amount of urea remains at the level of that in the intact mice. In the blood plasma of mice with the Lewis carcinoma the NSE suspension and nanocomplex reduce activity of aspartate aminotransferase, the basic marker of necrosis of the heart tissue, growth of which was caused by the tumour development. Doxorubicinum increases activity of alanine aminotransferase, the marker of the liver lesion; introduction of NSE in the nanocomplex composition prevents the growth of the enzyme activity. N-stearoylethanolamine, both in the nanocomplex and in suspension, modulates activity of enzymes of antioxidantive protection of the heart, kidney and liver tissue of mice with the Lewis carcinoma.

[The influence of N-stearoylethanolamine on the activity of antioxidant enzymes and on the level of stable NO metabolites in the rat testes and blood plasma at the early stages of streptozotocine-induced diabetes].

The influence of N-stearoylethanolamine was investigated on the activity of enzymes of antioxidant protection and content of stable metabolites of nitric oxide (NO) in the testes and plasma of rats at the early stages of development of streptozotocine-induced diabetes mellitus. It was shown that the activity of superoxide dismutase, catalase is reduced in the plasma and testes of animals with streptozotocin-induced (50 mg/kg) diabetes (blood glucose 8-10 mmol/L). A significant increase in the amount of nitrite and nitrate anions was revealed in the plasma of rats, while only the level of nitrite was significantly changed in the testes of animals. The per os administration of the NSE aqueous suspension in a dose of 50 mg/kg during 10 days to the rats with induced diabetes contributed to the normalization of catalase activity in the testis, which correlated with a decrease in the amount of TBA-reacting products and activity of superoxide dismutase and catalase in the blood plasma of animals; the use of NSE also contributed to the reduction of nitrite content in the gonads and to normalization of both nitrite and nitrate in the blood plasma of rats. The NSE administration to intact animals caused an increase in superoxide dismutase activity and significantly reduced the content of stable NO metabolites in the blood plasma of animals.

[Antioxidative effect of the N-stearoylethanolamine in the heart tissue and blood plasma of rats under doxorubicin treatment].

The influence of N-stearoylethanolamine on the alterated antioxidant enzyme activity in the heart tissue and blood plasma of rats under the doxorubicin treatment was investigated. It was shown that doxorubicin administration caused the decrease of antioxidant enzymes activity (superoxide dismutase and glutathione peroxidase) in the heart tissue. Administration of the NSE promoted the partial normalization of these enzymes activity. It was shown that doxorubicin treatment caused the increase of the urea and creatinine level in the blood plasma of experimental animals. The NSE administration normalized the level of the urea and did not affect creatinine level.

[Protective effect of N-stearoylethanolamine in acute alcohol intoxication in rats].

The influence of N-stearoylethanolamine on the nitric oxide system, the state of lipid peroxidation, antioxidant enzyme activity, content of phospholipids and fatty acids were studied under the acute ethanol intoxication (2.5 g/kg) in rats. The results of investigations show that acute ethanol intoxication caused abnormalities of the oxidative homeostasis accompanied by the accumulation of thiobarbituric acid reactive substances (TBARS). High catalase and glutathione peroxidase activity was also shown. The altered content of total and individual fatty acids of phospholipids in the rat liver tissue was found. The content of saturated fatty acids (palmitic, stearic) increased and amount of unsaturated (palmitoleic, oleic, linolenic) acids decreased under acute ethanol intoxication. The changes of nitric oxide content was found in the brain, plasma and red blood cells. N-stearoylethanolamine (NSE) in a dose of 50 mg/kg of body weight shows the pronounced antioxidative and hepatoprotective properties under these conditions. It was found that the preliminary NSE administration to rats inhibited accumulation of TBARS and caused a simultaneous increase of antioxidant enzyme activity. The NSE administration modulated also the content of total and individual fatty acids of phospholipids and the amount of nitric oxide in pathologically altered tissues. These results suggested that NSE protected the structural integrity and functional ability of cell membranes under the acute ethanol intoxication.

[The modelling of N-stearoylethanolamine effect of 17beta-estradiol in the organism of male rats].

The influence of N-stearoylethanolamine (NSE) on total 11-hydroxycorticosteroids (11-HCS) and testosterone level in the blood of male rats in normal conditions and under the action of 17beta-estradiol (400 mkg/kg of body weight during 3 days) was studied. It was shown that NSE administration per os (50 mg/kg of body weight during 7 days) to intact animals did not change the level of 11-HCS and of testosterone. The administration of NSE to estrogenized male rats decreased the elevated level of 11-HCS and normalized the amount of testosterone in blood. The correction of alterated weight of adenohypophysis and testis of estrogenized male rats compared to control can be a direct evidence of NSE-mediated modelling of the effect on hypothalamic-pituitary hormone system. The effect of NSE in the testis of estrogenized male rats inhibited the process of lipid peroxidation, caused the decrease of the amount of thiobarbituric acid reactive substances and increased the activity of superoxide dismutase and catalase. The NSE showed more expressed antioxidative effect compared to vitamin E. Taking into consideration all above mentioned data we suggested that NSE administration to male rats protected Leydig cells from damage under the increase of estrogen level.

[Anti-inflammatory effect of N-stearoylethanolamine in experimental burn injury in rats].

The biochemical mechanisms of anti-inflammatory effect of endocannabinoid congener N-stearoylethanolamine (NSE) was studied on the model of experimental burn in rats. The animals after the thermal burn of the skin received per os during 7 days the water suspension of NSE in a doze 10 mg/kg of body weight. In the other groups of rats the suspension was applied to the wound (the concentration of NSE was 10 mg/ml). It was shown for the first time that NSE accelerated the process of burn wound healing by the inhibition of proinflammatory cytokines (TNFalpha, IL-6) production. NSE caused the normalization of the iNOS and cNOS activity and of nitrite content in plasma, erythrocytes, liver and spleen of rats. NSE also modified the antioxidant enzymes (catalase, superoxide dismutase and glutathione peroxidase) activity and diminished the level of lipid peroxidation. The discovered anti-inflammatory NSE properties suggest the possibility of its usage for burn treatment.

[Immunosuppressive characteristics of N-stearoylethanolamine a stable compound with cannabimimetic activity].

Results of investigation of biochemical mechanisms of N-stearoylethanolamine (NSE) influence on the processes of allergic responses of immediate and delayed type (anaphylactic shock in guinea pigs and contact hypersensitivity to 2,4-dinitrochlorobenzene in mice) are presented in the paper. NSE was given per os during two weeks. It was found that in anaphylactic animals, NSE prevented the growth of histamine levels in the heart, kidneys and spleen, suppressed NO2(-) level increase in these organs and promoted its normalization. At the same time NSE prevented the decrease of the level of stable metabolite of nitrogen oxide - nitrite-anion (NO2(-)) in the liver and to a lesser degree in the lungs, and also decreased the activity both inducible and constitutive NO-synthases. NSE normalized the content of TBA-reactive products in the lungs and decreased it in the heart, diminished the decline of activity of glutathione peroxidase, catalase and superoxide dismutase. Effects of NSE depended on its daily dose. About 70% of animals which received NSE in a dose 65 mg/kg of body weight had no fatal outcome after the induction of anaphylactic shock. NSE suppressed the delayed type hypersensitivity response and normalized NO2(-) content in the blood plasma of mice but only at the dose of 50 mg/kg of weight. In the thymus of sensitized mice NSE diminished the content of NO2(-). Thus, though NSE has no affinity for specific CB receptors, in other words, it is not a typical endocannabinoid, its ability to influence the immediate and delayed type allergic reactions opens a perspective for creation of new medications which differ principally from existing pharmacological drugs with anti-allergic and immunosuppressive properties.

Long-term inhibition of hepatitis B virus in transgenic mice by double-stranded adeno-associated virus 8-delivered short hairpin RNA.

RNA interference (RNAi) was reported to block hepatitis B virus (HBV) gene expression and replication in vitro and in vivo. However, it remains a technical challenge for RNAi-based therapy to achieve long-term and complete inhibition effects in chronic HBV infection, which presumably requires more extensive and uniform transduction of the whole infected hepatocytes. To increase the in vivo transfection efficiency in liver, we used a double-stranded adeno-associated virus 8-pseudotyped vector (dsAAV2/8) to deliver shRNA. HBV transgenic mice were used as an animal model to evaluate the inhibition effects of the RNAi-based gene therapy. A single administration of dsAAV2/8 vector, carrying HBV-specific shRNA, effectively suppressed the steady level of HBV protein, mRNA and replicative DNA in liver of HBV transgenic mice, leading to up to 2-3 log(10) decrease in HBV load in the circulation. Significant HBV suppression sustained for at least 120 days after vector administration. The therapeutic effect of shRNA was target sequence dependent and did not involve activation of interferon. These results underscore the potential for developing RNAi-based therapy by dsAAV2/8 vector to treat HBV chronic infection, and possibly other persistent liver infections as well.

[Effect of N-stearoylethanolamine on the lipid peroxidation process and lipid composition of the rat liver in acute morphine intoxication].

The effect of N-stearoylethanolamine (NSE) on the lipid peroxidation process, antioxidant enzymes activity, phospholipid and fatty acid content in the rat liver tissues under acute morphine administration was studied. It was shown that morphine administration (30 mg/kg of body weight) caused an increase of the amount of thiobarbituric acid reactive substances (TBARS), alteration of antioxidant enzymes activity, decrease the protein level, quantity of total lipids and phospholipids, phosphatidylcholine, cholesterol esters; altered the content of some individual fatty acids. NSE administration (50 mg/kg of body weight) promoted normalization of the antioxidant enzymes activity and prevented the TBARS accumulation and decreased the total lipid and phospholipid quantity, increased the content of free and total cholesterol, corrected the level of free and individual fatty acids. It was assumed that NSE possessed antioxidative, membranoprotective and adaptive properties.

[Effect of flogensim in the treatment of postischemic inflammation (according to results of using interleukin-6 in patients with acute brain infarction)].

I1-6 in blood serum 109 patients with ischemic stroke was tested on 1st and 7 day after developing the disease. The decrease in concentration of I1-6 on 7 day was found after a complex therapy with Flogensim in the study group in comparison with the control group where a traditional therapy was used. The authors found considerable difference in consequences of the ischemic stroke on 21 day among patients pertaining to different group: the number of patients of the study group where results were better increases and number of the patients of this group with no dynamic or even worsening in neurological status decreases.

[Neuroprotective effect of N-acylethanolamines in chronic morphine dependence. II. Effect on the rat brain fatty acid composition].

The effect of N-acylethanolamines mixture (NAE) with saturated and unsaturated acyl chains on the fatty acid composition of the rat brain under chronic morphine dependence was investigated. Long-term administration of NAE reduced morphine-induced decrease of mono- and polyunsaturated fatty acids in the rat brain crude lipid extract. Furthermore, NAE restored the acyl chain spectrum, especially the content of docosahexaenoic acid in essential phospholipids--phosphatidylcholine and phosphatidylethanolamine. Pharmacological activity of NAE depended on a dose.

[Neuroprotective effect of N-acylethanolamines in chronic morphine dependence. III. Influence on the content of neurotransmitters in the rat brain].

The effect of the mixture of saturated and unsaturated N-acylethanolamines (NAEs) on the functional activity of catecholamine- and serotoninergic systems of the rat brain with experimental morphine dependence was investigated. A significant decrease of dopamine levels was found in the hypothalamus, midbrain and cortex of rats with morphine dependence. The administration of NAEs to rats with morphine dependence in time course dose of 35 mg/kg markedly increased the levels of dopamine in the hypothalamus, middle brain and cortex. Simultaneously the significant decrease of serotonine content was observed in the midbrain and cortex. The results obtained suggest that one of the important aspects of neuroprotective action of NAEs under morphine dependence is the restoration of dopamine content in the brain.