Peripheral Blood Leukocytes Of Patients Research Articles

Altered counts and mitochondrial mass of peripheral blood leucocytes in patients with chronic hepatitis B virus infection.

Hepatitis B virus (HBV) damages liver cells through abnormal immune responses. Mitochondrial metabolism is necessary for effector functions of white blood cells (WBCs). The aim was to investigate the altered counts and mitochondrial mass (MM) of WBCs by two novel indicators of mitochondrial mass, MM and percentage of low mitochondrial membrane potential, MMPlow%, due to chronic HBV infection. The counts of lymphocytes, neutrophils and monocytes in the HBV infection group were in decline, especially for lymphocyte (p = 0.034) and monocyte counts (p = 0.003). The degraded MM (p = 0.003) and MMPlow% (p = 0.002) of lymphocytes and MM (p = 0.005) of monocytes suggested mitochondrial dysfunction of WBCs. HBV DNA within WBCs showed an extensive effect on mitochondria metabolic potential of lymphocytes, neutrophils and monocytes indicated by MM; hepatitis B e antigen was associated with instant mitochondrial energy supply indicated by MMPlow% of neutrophils; hepatitis B surface antigen, antiviral therapy by nucleos(t)ide analogues and prolonged infection were also vital factors contributing to WBC alterations. Moreover, degraded neutrophils and monocytes could be used to monitor immune responses reflecting chronic liver fibrosis and inflammatory damage. In conclusion, MM combined with cell counts of WBCs could profoundly reflect WBC alterations for monitoring chronic HBV infection. Moreover, HBV DNA within WBCs may be a vital factor in injuring mitochondria metabolic potential.

Xiaoyao Pills, a Chinese patent medicine, treats mild and moderate depression: A randomized clinical trial combined with DNA methylation analysis

BackgroundXiaoyao pills (XYP) is a commercial Chinese patent medicine used in the treatment of depression. However, the mechanisms underlying its therapeutic effects, as well as the patients who can benefit from XYP, have not been evaluated so far. ObjectivesTo this end, we conducted a double-blinded, random, and placebo-controlled clinical trial of orally administered XYP in patients with depression. MethodsThe 17-item Hamilton Depression Rating Scale (HAMD-17) scores were recorded at baseline, and every 2 weeks after the start of treatment. To further elucidate the epigenetic mechanism of XYP, we performed mRNA sequencing and genome-wide DNA methylation sequencing using peripheral blood leukocytes of patients and healthy. ResultsXYP effectively alleviated the symptoms in patients with mild or moderate depressive disorders, particularly that of psychomotor retardation. XYP restored aberrant gene expression and DNA methylation patterns associated with depression, and the normalization of DNA methylation correlated with downregulation of several genes. In addition, altered DNA methylation levels in the XYP-treated samples were attributed to increased expression of the DNA methyltransferase DNMT1. ConclusionsOur study provides new insights into the epigenetic mechanism underlying depression and the therapeutic effects of XYP, along with an experimental basis for using XYP in the treatment of depression. Trial Registration InformationThe name of the registry and number: U.S. Clinical Trials Registry. The link to the registration: ClinicalTrials.gov ISRCTN12746343 (https://www.isrctn.com/ISRCTN12746343). The name of the trial register is “Efficacy and safety of the Xiaoyao pill for improving the clinical symptoms of stagnation of liver qi (chi) and spleen deficiency”. The clinical trial registration number is ISRCTN12746343.

Expression level of long non-coding RNA MALAT1, GAS5, DANCR and TUG1 in peripheral blood leukocytes of patients with non-alcoholic fatty liver disease

Purpose: Comparative analysis of the expression level of long non-coding RNAs MALAT1, GAS5, DANCR, TUG1 in peripheral blood leukocytes (PBL) of healthy people and patients with NAFLD (liver steatosis, NASH of varying activity, liver cirrhosis). Materials and methods: We examined 106 patients diagnosed with NAFLD for the first time: 31 patients with liver steatosis (LS), 64 patients with weak (WA), moderate (MA) and high (HA) NASH activity and 11 patients at the stage of liver cirrhosis (LC). The control group consisted of 30 healthy donors. The mRNA level of the TUG1, DANCR, MALAT1, GAS5 genes in PBL was determined by RT-PCR. Results: A higher level of expression of the TUG1 gene was registered in the PBL of patients with NASH-WA compared to LS, and a tendency was revealed to increase the level of TUG1 mRNA in the PBL with increasing NASH activity, which indicates the possibility of using the level of TUG1 expression in the PBL as a minimally invasive diagnostic (to distinguish between LS and NASH-WA) and a prognostic marker (with the progression of NAFLD). Analysis of the expression level of lncRNA MALAT1 showed no significant differences between all studied groups. Results were obtained indicating complex dynamics of the GAS5 expression level: the level of transcripts increases during the formation of liver steatosis and then decreases during the transition to NASH. It was shown that the level of DANCR expression in the PBL of patients with NASH-WA is significantly lower than in patients with liver steatosis and NASH-MA. Conclusion: New data were obtained on the expression level of the MALAT1, GAS5, DANCR, TUG1 lncRNAs in the PBL of patients with NAFLD, indicating the possibility of using the level of TUG1 expression in the PBL as a minimally invasive diagnostic and prognostic marker in NAFLD. It has also been shown that the level of DANCR mRNA in PBL may have some diagnostic value in distinguishing between LS and NASH-WA.

Blood Level of Nitric Oxide Metabolites and Expression of Genes Regulating NO Synthesis in Early Forms of Non-Alcoholic Fatty Liver Disease.

The levels of NO metabolites in the plasma and mRNA of the NOS3, ATG9B, and NOS2 genes in peripheral blood leukocytes of healthy people and patients with early forms of non-alcoholic fatty liver disease (steatosis and weak activity non-alcoholic steatohepatitis) were studied. In patients with steatohepatitis, the concentration of NO metabolites in the blood and the level of mRNA of the NOS2 gene were higher than in patients with steatosis and healthy people. These differences can be of diagnostic value for distinguishing between steatosis and weak activity steatohepatitis in non-alcoholic fatty liver disease. A correlation between the levels of NO metabolites and the expression of the NOS2 gene in weak activity steatohepatitis was established, which indicates activation of NO synthesis in non-alcoholic steatohepatitis due to the expression of the inducible NO synthase gene. The level of the NOS2 gene mRNA in peripheral blood leukocytes of patients with weak activity steatohepatitis correlated with the level of TNFα and IL-6 cytokines. An increase in the level of NO in the blood in weak activity steatohepatitis correlated with the level of MDA, an indicator of oxidative stress.

Potential role of mitochondrial dysfunction in arrhythmogenesis in coronary artery disease

Introduction. Coronary artery disease (CAD) continues to be the most common pathology in the structure of cardiovascular diseases over the past decades, both in Russia and around the world. In the normal condition, the mitochondria of all body cells have the same function capabilities due to the carriage of the same genome. Therefore, it is possible to assess the respiration activity of cardiomyocyte mitochondria by the respiration of mitochondria from peripheral blood leukocytes.Aim: To compare respiratory activity of mitochondria of peripheral blood leukocytes in patients diagnosed with coronary artery disease and coronary artery disease with developed cardiac rhythm disorders (CRD).Material and methods. The studied groups included 45 patients with CAD without CRD and 39 patients with CAD complicated by CRD. Mitochondria were isolated from peripheral blood leukocytes by differential centrifugation. The rate of oxygen loss in pyruvate-malate and succinate incubation buffers was measured when isolated mitochondria were introduced, as well as when palmitic acid was added to the medium. Oxygen consumption rate for the V3 (active phosphorylating) and V4 (nonphosphorylating) metabolic sates was determined, and on their basis respiratory control coefficient was calculating using the formula V3/V4. Statistical data processing was carried out using STATISTICA 13.0 software.Results. Oxygen consumption rate in mitochondria of patients with uncomplicated CAD and CAD with CRD had no significant differences in either pyruvate-malate or succinate buffers. When palmitic acid was added to the incubation medium, the mitochondria of CAD patients without CRD significantly increased oxygen consumption rate in both incubation media. Mitochondria of CAD patients with CRD did not change oxygen consumption rate in both metabolic states after the addition of palmitic acid in incubation media.Conclusion. On the basis of the data obtained, it can be concluded that the function capabilities of mitochondria in the complicated course of CAD has been exhausted, which manifests itself in the inability to increase ATP synthesis in response to the introduction of additional substrates.

Нейтрофильные синцитии в периферической крови пациентов с коронавирусной инфекцией (COVID-19)

Introduction. COVID-19, an infectious disease etiologically related to the coronavirus SARS-CoV-2, manifests with fever, respiratory symptoms, and lymphocytopenia. Pathogenic mechanisms of COVID-19 remain largely unclear. SARS-CoV-2 infection was found to cause the formation of multinucleated syncytia, which dominate in lung tissues and can target lymphocytes, potentially contributing to lymphocytopenia. The formation of these structures is considered to be the result of the fusogenic ability of the SARS-CoV-2 spike-like protein. The aim was to study the morphology and pathogenic significance of intercellular interactions of peripheral blood leukocytes in patients with coronavirus infection caused by SARS-CoV-2. Materials and methods. We studiedblood samples of 30 patients infected with the SARS-CoV-2 virus, who underwent treatment in the Regional Clinical Hospital No. 2, Vladivostok, in 2021–2022. The COVID-19 diagnosis was confirmed using a PCR test. Peripheral blood buffy coat samples fixed in OsO4 were embedded in white resin epoxy (Sigma-Aldrich, USA), and then ultrathin sections were prepared. Results. Against the background of the morphological leukocyte abnormality, in the peripheral blood of patients with COVID-19, neutrophilic syncytia were formed. These syncytia consisted of cells interconnected by cytoplasmic bridges. We distinguished three stages of the neutrophilic syncytium formation, at the first one, cells approaching each other and forming large outgrowths involved in cell docking. The second one was characterized by the formation of a synaptic connection between cells (split contact). At the last stage, outer cell membranes fused completely and the syncytium formed. Conclusion. Based on our analysis we assume that the formation of neutrophil syncytia in the peripheral blood of patients with COVID-19 creates conditions for hematogenous dissemination of SARS-CoV-2 and viremia, generalization of infection and the development of multiorgan pathology. Keywords: blood, leukocytes, neutrophil syncytia, morphology, COVID-19, coronavirus, SARS-CoV-2

Surgical resection of lung cancer inhibits mRNA expression of GOT2 gene encoding kynurenine aminotransferase in leukocytes.

Lung cancer is the most common malignant tumour. More than 80% of all diagnosed cases are non-small cell carcinoma which can be effectively treated by radical resection. Despite significant progress in the field of diagnostic and therapeutic methods, the results of lung cancer treatment are still unsatisfactory. Lung cancer is detected relatively late, which leads to an unfavourable prognosis. Kynurenine aminotransferases are an important element of the kynurenine pathway of tryptophan metabolism, which has recently aroused great interest from the aspect of possible use as a target point of personalized therapies in malignant tumours.The aim of the study was to analyze the expression of the selected gene of kynurenine aminotransferases GOT 2 at the mRNA level in peripheral blood leukocytes of patients with lung cancer. The mRNA expression of the GOT 2 gene was tested on blood samples from 50 patients treated surgically for non-small cell lung cancer.The control group consisted of 15 healthy individuals.The determination of mRNA expression of the GOT 2 gene was performed using the real-time PCR method.The GAPDH gene was used as the endogenous reference level. The mRNA expression of the GOT2 gene on the 6th day after surgery was statistically significantly lower than before surgery (p = 0,05). In the study group, the average LogRQ mRNA expression of the GOT2 gene before the procedure was 0.192082±0.292174 in woman. This was statistically significantly higher than in men whose average LogRQ mRNA expression of the GOT2 gene before the procedure was 0.004210±0.235065 (p=0.0183). Surgical resection of lung cancer results in inhibition of GOT2 mRNA expression in leukocytes. Further studies are expected to show whether it may be used as a target point for personalized therapies in lung cancer.

Expression of transient receptor potential channels on peripheral blood leukocytes of patients with chronic obstructive pulmonary disease

It is known that transient receptor potential (TRP) channels are receptors for reactive oxygen species and components of cigarette smoke, and therefore may be involved in the pathogenesis of chronic obstructive pulmonary disease (COPD).Aim: To investigate the expression of TRPV1, TRPV4, TRPM8 and TRPA1 channels on peripheral blood leukocytes of COPD patients and healthy individuals and to establish its possible relationships with inflammatory markers and lung function parameters.Methodology and Research Methods. 23 patients with COPD of varying severity and 17 individuals without bronchial obstruction were examined. The expression of TRP channels on monocytes, lymphocytes and granulocytes was determined by indirect flow cytometry. Plasma concentrations of cytokines were measured in a multiplex assay. Lung function parameters were assessed by spirometry.Results. Monocytes from COPD patients were characterized by increased expression of TRPV1 (98.8 (97.6; 99.6)% vs. 97.8 (96.3; 98.6)%, p = 0.03) and TRPA1 (99.2 (98.7; 99.8)% vs. 95.1 (94.2; 98.4)%, p = 0.003). In addition, COPD was associated with an increased ratio of TRPV4/TRPM8 expression on monocytes (0.66 (0.51, 0.90) vs. 0.27 (0.12, 0.70), p = 0.02) and granulocytes (0.97 (0.86; 0.92) vs. 0.29 (0.06; 0.89), p = 0.003). TRPV4 expression on monocytes showed correlation with CXCL10 level (ρ = 0.55, p = 0.01), and for TRPM8 inverse relationships were found with the concentrations of IL-6 (ρ = –0.56, p = 0.02) and IL-10 (ρ = –0.55, p = 0.02). We did not find any associations between the expression of TRP channels and lung function parameters.Conclusion. Increased expression of TRPV1 and TRPA1, as well as an increased TRPV4/TRPM8 ratio on peripheral blood monocytes of COPD patients may indicate the contribution of these receptors to the proinflammatory activation of the cells and indicate possible prospects of TRP channels pharmacological modulation for the purpose of the disease therapy.

THU235 Usefulness Of Digital Droplet Polymerase Chain Reaction (ddPCR) For Molecular Diagnosis Of McCune-Albright Syndrome In DNA From Peripheral Blood Leukocytes

Abstract Disclosure: A.G. de Faria: None. L.R. Montenegro: None. A.A. Jorge: None. R.S. Jallad: None. R.M. Martin: None. M.C. Fragoso: None. A.P. Canton: None. C.E. Seraphim: None. F.R. Tinano: None. N.C. Pinto: None. B.B. Mendonca: None. A. Latronico: None. V.N. Brito: None. Background: McCune-Albright Syndrome (MAS) is a rare congenital disorder caused by post-zygotic activating mutations in GNAS gene. Due to the mosaic pattern of this disease, mutation abundance is frequently low in several tissues, including blood cells. The emergence of digital droplet Polymerase Chain Reaction (ddPCR) is a breakthrough technology of quantitative PCR useful for a targeted detection and quantification of rare events. Objective: This study aims to identify the GNAS mutation in peripheral blood leukocytes of patients with MAS using ddPCR. Patients and Methods: Thirty-four patients (25 female and 9 male) with typical (classic triad) or atypical (one or two features) MAS were included. DNA was extracted from peripheral blood and was analyzed through ddPCR method. Two ddPCR mutation detection assays were used, each one targeting the p.R201C or p.R201H mutations. For each assay, the Taqman PCR system was used with two fluorescence probes: the first labeled with HEX, targeting de (WT allele), and the second labeled 6-FAM, targeting the (mutated allele). The mixture (reagents + DNA) was divided into 20,000 nanodrops, which was placed on a PCR plate and amplified by PCR. After amplification by PCR, the fluorescence of each droplet was read on the 200 Droplet Digital PCR system (Bio-Rad), being classified as positive (drop containing the target sequence) or negative. Results were analyzed using Quantasoft software (Bio-Rad) to determine the total amount of positive droplets in the original sample. The results were reported in fractional abundance (FA), corresponding to the percentage of mutated alleles within the total alleles. Results: Among all patients, the most common MAS feature was osteofibrous dysplasia (OFD) (73.5%), followed by peripheral precocious puberty (PPP) (64.7%), and café-au-lait skin spots (52.9%). Acromegaly or hyperthyroidism was diagnosed in 6 and 4 patients, respectively. Classical MAS triad, 2 clinical features, or 1 clinical feature (PPP or OFD) were present in 15, 9, and 10 patients, respectively. GNAS mutations were identified in 12/34 (35.3%) MAS patients: the p.R201C in 5 and the p.R201H in 7. Among them, 8 patients presented typical MAS and 4 patients with two MAS clinical features, respectively. Considering only typical MAS group, the positive molecular test rate was 53.3% (8/15). GNAS mutations were not identified in patients with only one MAS feature. There was a positive correlation between a positive molecular test and the number of MAS clinical features (p = 0.01). Median FA was 1.45%, ranged from 0.25% to 8.4%. There was no association between FA rate and the number of MAS clinical features (p=1.0). Conclusion: ddPCR representeds a more sensitive tool for identifying GNAS mutations in peripheral blood leukocytes, even with low abundance, in patients with classical and atypical MAS. Presentation: Thursday, June 15, 2023

Study of the Association of Polymorphic Variants rs2295080 and rs1883965 of the <i>MTOR</i> Gene with the Development of Pulmonary Sarcoidosis

The aim of the study was to study the association of polymorphic variants rs2295080 and rs1883965 of the MTOR gene with the risk of developing pulmonary sarcoidosis. The study included 253 people (122 patients diagnosed with morphologically verified sarcoidosis with lung involvement (average age – 41.00 ± 12.56 years) and 131 healthy people (control group) (average age – 44.00 ± 14.23). The distribution of alleles and genotypes of polymorphic markers rs2295080 and rs1883965 of the MTOR gene was analyzed in the study groups. There was a statistically significant increase in the level of mRNA expression of the MTOR gene in PBL of patients with pulmonary sarcoidosis compared with the control group (p = 0.007). A decrease in the number of transcripts of this gene was noted in patients receiving therapy compared with patients without therapy (p = = 0.025). There were no statistically significant differences in the distribution of allele and genotype frequencies for polymorphic markers rs2295080 and rs1883965 of the MTOR gene in the group of patients with pulmonary sarcoidosis and in the control group: (χ2 = 0.196, d.f. = 1, p = 0.658 and χ2 = 0.637, d.f. = 2, p = = 0.728) and (χ2 = 0.034, d.f. = 1, p = 0.855 and χ2 = 0.051, d.f. = 2, p = 0.975) respectively. Conclusion: аn increased level of expression of the MTOR gene in peripheral blood leukocytes of patients with pulmonary sarcoidosis may indicate the involvement of this gene in the pathogenesis of this disease. Polymorphic markers rs2295080 and rs1883965 of the MTOR gene are not associated with the risk of developing pulmonary sarcoidosis. Probably, an increase in the expression level of the MTOR gene in patients with pulmonary sarcoidosis is due to the development of inflammation.

The state of mitochondrial membrane potential in peripheral blood leukocytes of patients with chronic obstructive pulmonary disease

Introduction. Mitochondria provide energy homeostasis of the cell by maintaining an optimal transmembrane electrochemical gradient (ΔΨm), which does not allow excessive formation of reactive oxygen species (ROS). However, under conditions of pathology, the normal functioning of mitochondria is disrupted, which can lead to ATP deficiency and/or increased production of ROS.Aim. The aim of this study was to investigate the ΔΨm parameters and their relationship with the expression of TRP channels in peripheral blood leukocytes of patients with chronic obstructive pulmonary disease (COPD).Materials and methods. The study included 23 patients with COPD of varying severity, 8 smokers without signs of bronchial obstruction and 9 healthy volunteers who had never smoked. All subjects underwent spirometry to assess the lung function. ΔΨm was determined by staining the cells with tetramethylrhodamine ethyl ester (TMRE) and measuring the fluorescent signal by flow cytometry, under basal conditions and pro-inflammatory stimulation with phorbol-12-myristate-13-acetate (PMA).Results. We found that COPD patients were characterized by a significant increase in basal ΔΨm of monocytes (161.8 [153.8; 206.8] vs. 129.3 [75.5; 161.8], p=0.03) and lymphocytes (209,7 [184.7; 257.8] vs. 122.5 [67.9; 164.3], p=0.003) as compared with the control group. Stimulation of cells with PMA led to multidirectional changes in ΔΨm, while its increased level was still preserved in COPD. In monocytes of COPD patients, a decrease in ΔΨm in response to PMA stimulation was prevalent (75%), while in the majority (53.9%) of individuals in the control group ΔΨm, on the contrary, increased (p=0.08). In addition, among COPD patients, an increase in ΔΨm in monocytes was accompanied by an enhanced expression of TRPV4, while in the control group, among individuals with positive dynamics of ΔΨm, TRPV4 expression was, on the contrary, reduced.Conclusion. The increased level of ΔΨm in the mononuclears of COPD patients is consistent with previously detected enhanced ROS production, but does not support the assumption about energy deficit in the cells. The revealed differences in the relationship between TRPV4 expression and ΔΨm dynamics may indicate the presence of pathological features in TRP signaling in COPD patients.

Decreased expression of CD200 on peripheral blood leukocytes in alcohol dependence

Chronic alcohol use induces innate immune genes, which activate the innate immune system. Neuroimmune regulatory proteins [e.g., Cluster of Differentiation 200 (CD200)] are immune response regulators and are involved in balancing the immune response. This study aimed to investigate the expression of CD200 on the surface of peripheral blood leukocytes in patients with alcohol use disorder and compare them with controls. Fifty male patients with alcohol use disorder were included in the study. A baseline assessment was done, and alcohol use history, craving, and withdrawal scores were collected. A 2-mL venous blood sample was collected from cases and controls for immunophenotyping of CD200. The control group consisted of 50 participants with similar socio-economic backgrounds. The cellular expression of CD200 on total leukocytes (median ± IQR) [39.94 (28.85, 50.01)] in cases was significantly lower compared to controls [45.07 (37.70, 51.69)] (U = 896, p = 0.015). Expression of CD200 on lymphocytes in cases was negatively correlated with years of heavy drinking and this was statistically significant (r = −0.321, p = 0.023). The study indicates that cellular expression of CD200 on the surface of peripheral blood leukocytes is reduced in alcohol-dependent patients. This reduction can contribute to exaggerated immune activity, release of pro-inflammatory cytokines, chronic microglial activation, neuroinflammation, and neurodegeneration in alcohol dependence.

Association of IFIH1 and DDX58 genes polymorphism with susceptibility to COVID-19.

Pattern recognition receptors of the innate immune system, such as RIG-I and MDA5, are responsible for recognizing viruses and inducing interferon production. Genetic polymorphisms in the coding regions of RLR may be associated with the severity of COVID-19. Considering the contribution of the RLR signaling in immune-mediated reactions, this study investigated the association between three SNP in the coding region ofIFIH1andDDX58genes with the susceptibility to COVID-19 in the Kermanshah population, Iran. 177 patients with severe and 182 with mild COVID-19 were admitted for this study. Genomic DNA was extracted from peripheral blood leukocytes of patients to determine the genotypes of two SNPs, rs1990760(C>T) and rs3747517(T>C)IFIH1gene and rs10813831(G>A)DDX58gene using PCR-RFLP method. Our results showed that the frequency of the AA genotype of rs10813831(G>A) was associated with susceptibility to COVID-19 compared to the GG genotype (p = 0.017, OR = 2.593, 95% CI 1.173-5.736). We also observed a statistically significant difference in the recessive model for SNPs rs10813831 variant (AA versus GG + GA,p = 0.003, OR = 2.901, 95% CI 1.405-6.103). Furthermore, No significant association was found between rs1990760 (C>T) and rs3747517(T>C) ofIFIH1gene polymorphisms with COVID-19. Our findings suggest thatDDX58rs10813831(A>G) polymorphism may be associated with COVID-19 severity in the Kermanshah population,Iran.

Oxidative stress in peripheral blood leukocytes of patients with chronic obstructive pulmonary disease

Introduction.It is known that oxidative stress causes pathological changes in the tissues and organs of the human body and in many cases it can explain the disorders associated with chronic obstructive pulmonary disease (COPD).Aim.The aim of the study was to investigate the degree of oxidative stress in peripheral blood leukocytes of COPD patients.Materials and methods. We examined 23 patients with COPD of varying severity, 8 smokers without signs of bronchial obstruction and 9 healthy volunteers who had never smoked. All subjects underwent spirometry to assess lung function. The level of oxidative stress was determined using flow cytometry by adding 10 µM 2',7'-dichloro dihydrofluorescein diacetate to isolated peripheral blood leukocytes, in basal conditions and under pro-inflammatory stimulation with 0.1 ng/ml phorbol-12-myristate-13-acetate (PMA).Results.Patients with COPD demonstrated elevated levels of intracellular oxidative stress compared with controls both under basal conditions and during PMA stimulation (p<0.05). When analyzed in subgroups, stimulation led to a significant increase in oxidative stress regardless of cell type (p<0.01). Smokers tend to have elevated measured values, occupying an intermediate position between non-smokers and COPD patients. The absolute increase in the oxidative stress index was higher in the cells of COPD patients; the values of the relative increase were almost identical in the studied groups. The dynamics of oxidative stress in the cells of smokers tended to be more pronounced than in non-smokers.Conclusion.The obtained results indicate that the leukocytes of COPD patients are characterized by a higher level of intracellular oxidative stress. In addition, smoking has been found to be a factor of oxidative stress development in healthy individuals.

Baseline and oxidatively damaged DNA in end-stage renal disease patients on varied hemodialysis regimens: a comet assay assessment.

Global estimates exhibit that one million people have end-stage renal disease, a disease-state characterized by irreversible loss of kidney structure and function, thus necessitating renal replacement therapy. The disease-state, oxidative stress, inflammatory responses, as well as the treatment procedure can have damaging effects on the genetic material. Therefore, the present study was carried out to investigate DNA damage (basal and oxidative) using the comet assay in peripheral blood leukocytes of patients (n = 200) with stage V Chronic Kidney Disease (on dialysis and those recommended but yet to initiate dialysis) and compare it to that in controls (n = 210). Basal DNA damage was significantly elevated (1.13x, p ≤ 0.001) in patients (46.23 ± 0.58% DNA in tail) compared to controls (40.85 ± 0.61% DNA in tail). Oxidative DNA damage was also significantly (p ≤ 0.001) higher in patients (9.18 ± 0.49 vs. 2.59 ± 0.19% tail DNA) compared to controls. Twice-a-week dialysis regimen patients had significantly elevated % tail DNA and Damage Index compared to the non-dialyzed and to the once-a-week dialysis group implying dialysis- induced mechanical stress and blood-dialyzer membrane interactions as probable contributors to elevated DNA damage. The present study with a statistically significant power implies higher disease-associated as well as maintenance therapy (hemodialysis)-induced basal and oxidatively damaged DNA, which if not repaired has the potential to initiate carcinogenesis. These findings mark the need for improvement and development of interventional therapies for delaying disease progression and associated co-morbidities so as to improve life expectancy of patients with kidney disease.

Identification of hsa_circ_0001445 of a novel circRNA-miRNA-mRNA regulatory network as potential biomarker for coronary heart disease.

To evaluate the hsa_circ_0001445 level in peripheral blood leukocytes of patients with coronary heart disease (CHD) and its related clinical factors, and predict its circRNA-miRNA-mRNA regulatory network in CHD pathogenesis via bioinformatics analysis. Peripheral blood leukocytes were isolated from the whole blood samples of 94 CHD patients (aged 65.96 ± 9.78 years old) and 126 healthy controls (aged 60.75 ± 8.81 years old). qRT-PCR was used to quantify the expression level of circRNA and subsequently analyze its association with CHD clinical parameters. Via bioinformatics algorithm and GEO datasets, differential miRNA expression was evaluated using the Limma package. A miRNA-mRNA regulatory network was predicted by cyTargetLinker. ClusterProfiler was employed to perform functional enrichment analysis of the circRNA network to investigate its role in CHD pathogenesis. The expression of hsa_circ_0001445 in peripheral blood leukocytes of CHD patients was downregulated compared with that of healthy controls. Positive correlations were evident between hsa_circ_0001445 expression level and the levels of hemoglobin, triglycerides, high- and low-density lipoprotein cholesterol. A significant negative correlation was also found between hsa_circ_0001445 expression level and age and the neutrophil level. Low expression of hsa_circ_0001445 exhibited a discriminatory ability between CHD patients and healthy controls with a sensitivity of 67.5% and a specificity of 76.6% (p < 0.05). By bioinformatics analysis, 405 gene ontology terms were identified. The Kyoto Encyclopedia of Genes and Genomes terms focused principally on the PI3K-Akt signaling pathway. hsa_circ_0001445 was associated with the expression of three miRNAs that may regulate 18 genes involved in KEGG processes: hsa-miR-507, hsa-miR-375-3p, and hsa-miR-942-5p. The hsa_circ_0001445 level in peripheral blood leukocytes may serve as a biomarker for CHD diagnosis. Our work on circRNA-miRNA-mRNA networks suggests a potential role for hsa_circ_0001445 in CHD development.

Морфология лейкоцитов периферической крови у пациентов с новой коронавирусной инфекцией (COVID-19)

Introduction. The rapid spread of the COVID-19 pandemic caused by SARS-CoV-2 initially indicated a significant immune system involvement, including peripheral blood leukocytes. Hematologic changes have already been described in the first cases of COVID-19; however, only few studies reported morphological cell abnormalities in peripheral blood smears. The aim of the study was to characterize the morphology of peripheral blood leukocytes in patients with COVID-19 and determine the significance of its changes in the immune system dysfunction and for the disease diagnosis. Materials and methods. We studied blood samples of 30 patients infected by SARS-CoV-2 virus (confirmed with PCR tests) who were treated in Regional Clinical Hospital No. 2 in Vladivostok. Peripheral blood buffy coat samples fixed in osmium tetroxide were embedded in epoxy with subsequent preparation of semithin and ultrathin sections. Results. To the best of our knowledge, it is the first study to show nuclear pathological changes in neutrophils and monocytes in blood buffy coat of the patients with coronavirus infection with light and electron microscopy of peripheral blood. We found cells with abnormal nuclear shape and leukocyte apoptotic degeneration absent in healthy individuals. The identified morphological leukocyte abnormalities should be considered as signs of hyporeactivity of these cells, which indirectly indicate a decrease in their bactericidal potential in COVID-19. At the same time, the secretory degranulation of neutrophils with the release of the contents of the granules into the extracellular space was poorly visualized. We observed smooth plasmalemma of leukocytes with a small number of microvilli involved in phagocytosis and numerous apoptotic leukocytes with reduced phagocytic ability. Conclusion. The detection of morphological abnormalities of circulating leukocytes in patients with COVID-19 is of diagnostic and prognostic value in this pathology. Keywords: blood, neutrophils, monocytes, morphology, COVID-19, SARS-CoV-2, karyopathological changes

Эпигеномные изменения в лейкоцитах периферической крови у пациентов с гипертонической болезнью и дисциркуляторной энцефалопатией

В настоящее время эпигеномный ландшафт, лежащий в основе гипертонической болезни и дисциркуляторной энцефалопатии, мало изучен. Идентификация эпигенетического паттерна в комплексе с генетическим паспортом индивида может предоставить дополнительную информацию об этиологии заболеваний и риске развития осложнений, а также о выборе стратегии и тактики лечения. В представленной работе проведена количественная оценка уровня полногеномного метилирования ДНК и степени компактизации хроматина в лейкоцитах периферической крови у пациентов с гипертонической болезнью и дисциркуляторной энцефалопатией и здоровых добровольцев из Санкт-Петербурга. У пациентов с гипертонической болезнью выявлены гендерные различия в риске развития дисциркуляторной энцефалопатии. По сравнению с контролем при гипертонической болезни повышены уровень метилирования ДНК и степень компактизации хроматина. В исследованных группах у мужчин и женщин обнаружена различная эпигеномная реакция лейкоцитов на лекарственную терапию при данных заболеваниях. Currently, the epigenomic landscape underlying hypertension and dyscirculatory encephalopathy is poorly understood. Identification of the epigenetic pattern in combination with genetic passport can provide additional information about the etiology of diseases and the risk of complications, as well as the choice of strategy and tactics of treatment. We carried out a quantitative assessment of the level of genome-wide DNA methylation and the degree of chromatin compaction in peripheral blood leukocytes in patients with hypertension and dyscirculatory encephalopathy, and also in healthy volunteers from St. Petersburg. Gender differences in the risk of developing dyscirculatory encephalopathy were revealed in patients with hypertension. It was found that the level of DNA methylation and the degree of chromatin compaction are increased in hypertension (compared to control). In the studied groups, men and women showed different epigenomic reactions of leukocytes to drug therapy in these pathologies.

Hypofractionated vs. Conventionally Fractionated Radiotherapy for Glioblastoma: Peripheral Blood Leukocyte Comparison prior to and Following Chemoradiation

<h3>Purpose/Objective(s)</h3> The diagnosis of glioblastoma portends a poor prognosis with extremely low 5-year survival rates. Immune suppression related to the tumor, radiotherapy (RT), and chemotherapy has been documented following treatment and appears associated with poor tumor outcome. Previous study suggests radiotherapy contributes to lymphopenia, and that a shorter course of treatment may better spare the lymphocyte population. However, immunosuppressive monocytes, such as myeloid-derived suppressor cells, expand in number during treatment and may also affect tumor control directly and by contributing to lymphopenia. We analyzed clinical measures of peripheral blood leukocytes in patients receiving conventionally fractionated (CF) RT and hypofractionated (HF) RT for glioblastoma. We hypothesized monocyte quantities expand more with CF RT, compared to HF RT following concurrent chemoradiation. <h3>Materials/Methods</h3> We retrospectively reviewed records from patients treated for diagnosis of glioblastoma between 7/1/2011 and 6/2/2021 at our institution. Patients over the age of 18 who completed their glioblastoma RT course, had not received prior RT, and had sufficient lab data for comparison were included. Clinical peripheral blood counts were recorded prior to and following chemoradiation. Values were analyzed using descriptive summary statistics and t-confidence intervals. CF RT for newly diagnosed glioblastoma usually consists of approximately 6000 centigray (cGy) in 30 fractions. HF RT schemes used were generally 3400-4500 cGy in 10-15 fractions. <h3>Results</h3> Lab results from 80 patients (65 CF, 15 HF) were analyzed prior to and following RT. The median patient age was 59 years (Q1, Q3: 47, 66). 57% were male. All patients received concurrent temozolomide. Absolute leukocyte count decreased by 4.8 10⁹/L (3.7, 5.9) and 1.9 10⁹/L (-0.1, 3.8) for CF RT and HF RT, respectively. Absolute lymphocytes decreased by 7.0 10⁹/L (5.8, 8.1) in CF RT and 5.3 10⁹/L (3.5, 7.1) in HF RT, while the percent lymphocytes increased by 1.1% (-1.8%, 4.1%) for CF RT and 2.1% (-3.9%, 8.0%) for HF RT, respectively. Absolute monocytes decreased by 0.1 10⁹/L (0.0, 0.2) for CF RT and 0.1 10⁹/L (-0.1, 0.3) for HF RT. The percent monocytes increased by 2.7% (1.3%, 4.1%) in CF RT and by 0.2% (-2.2%, 2.6%) in HF RT. <h3>Conclusion</h3> We observed different effects in peripheral blood leukocyte populations in glioblastoma patients receiving CF RT and HF RT. Percent monocytes had the greatest increase of the white blood cell types in CF RT, while percent lymphocytes had the greatest increase after concurrent chemoradiation in those receiving HF RT. Further study will better define the effects of RT on the various cell populations important for anti-neoplastic immunity and assess impact on survival.

Investigation the importance of IL-6 gene polymorphism with the severity of infection in COVID-19 patients of Wasit Province, Iraq

Emerged coronavirus disease 2019 (COVID-19) was a pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-COV-2). Disease severity is associated with elevated levels of proinflammatory cytokines, such as interleukin-6 (IL-6). Genetic polymorphisms in the regulatory regions of cytokine genes may be associated with differential cytokine production in COVID-19 patients. This study aimed to investigate the association between potentially functional single-nucleotide polymorphisms (SNP) in the promoter region of IL-6 and the severity of susceptibility to COVID-19 in an Iraq population. In total, 120 individuals (40 patients with severe COVID-19, 40 patients with mild COVID-19 and 40 healthy people) were recruited for this cohort study. Genomic DNA was extracted from peripheral blood leukocytes of patients to determine the genotype of SNPrs1800795 (-174 G&gt;C) in the promoter region of the IL-6 gene by Sanger sequencing using ABI3730XL, automated DNA sequencer, by Macrogen Corporation – Korea after sent PCR products.