Peripheral Blood Mononuclear Leukocytes Research Articles

Sorafenib- Taurine Combination Model for Hepatocellular Carcinoma Cells: Immunological Aspects.

Sorafenib (Sor) is a multi-kinase inhibitor. It is recommended for the treatment of advanced hepatocellular carcinoma (HCC). However, Sor has severe and marked side effects. On the other hand, taurine (Tau) has been shown to enhance the therapeutic effects of cancer chemotherapy and also to enhance the function of leukocytes. Here, we aimed to investigate the enhancing efficacy of Sor as well as minimizing its marked side effects by using Tau in combination in an immunological aspect. We evaluated the influence of Sor and Tau combination on the expression pattern of FOXP3 gene in HepG2 cells compared to peripheral blood mononuclear leukocytes (PBMCs) as control normal cells. Also, the levels of TGF-β and IL-10 released in culture media of both cells were determined. Our results revealed that, Tau reduced cytotoxicity of Sor on PBMC indicated by lactic dehyrogenase (LDH) release assay. In addition, Sor-Tau combination led to FOXP3 down-regulation in hepatic cancer cells (HepG2). The results showed also that, TGF-β levels decreased significantly in their culture media. In contrary, the cytokine increased in PBMCs culture media. Moreover, IL-10 was significantly elevated in the culture media of both cells. This study could open new avenues for the improvement of therapeutic efficacy of Sorafenib treated HCC patients by using Tau in combination.

Establishment of Novel Cells Stably Secreting Various Human IL-18 Recombinant Proteins.

The immunotherapies against cancer, autoinmmune diseases or infection are remarkable development. These days programmed cell death (PD)-1 antibody-induced immune checkpoint blockade or chimeric antigen receptor-T cells (CAR-T) have been shown to have eminent therapeutic effects on tumor development. We have focused on adoptive transfer with human gamma delta T cells for novel immunotherapies. Additionally, IL-18 is one of the cytokines that enhances cytokine secretion and cytotoxicity of human gamma delta T cells. Thus, we established novel cell lines stably expressing and secreting various types of human recombinant IL-18 proteins to their culture supernatants using episomal vector. We also differentiated primary cultured human gamma delta T cells from peripheral blood mononuclear leukocytes to validate biological activity of the IL-18 proteins using measuring IFN-γ by ELISA. Finally, we demonstrated that the supernatant could activate human gamma delta T cells using monitoring interferon gamma in culture medium.

Rheumatoid arthritis reprograms circadian output pathways

ObjectiveWe applied systems biology approaches to investigate circadian rhythmicity in rheumatoid arthritis (RA).MethodsWe recruited adults (age 16–80 years old) with a clinical diagnosis of RA (active disease [DAS28 > 3.2]). Sleep profiles were determined before inpatient measurements of saliva, serum, and peripheral blood mononuclear leukocytes (PBML). Transcriptome and proteome analyses were carried out by RNA-SEQ and LC-MS/MS. Serum samples were analysed by targeted lipidomics, along with serum from mouse collagen induced-arthritis (CIA). Bioinformatic analysis identified RA-specific gene networks and rhythmic processes differing between healthy and RA.ResultsRA caused greater time-of-day variation in PBML gene expression, and ex vivo stimulation identified a time-of-day-specific RA transcriptome. We found increased phospho-STAT3 in RA patients, and some targets, including phospho-ATF2, acquired time-of-day variation in RA. Serum ceramides also gained circadian rhythmicity in RA, which was also seen in mouse experimental arthritis, resulting from gain in circadian rhythmicity of hepatic ceramide synthases.ConclusionRA drives a gain in circadian rhythmicity, both in immune cells, and systemically. The coupling of distant timing information to ceramide synthesis and joint inflammation points to a systemic re-wiring of the circadian repertoire. Circadian reprogramming in response to chronic inflammation has implications for inflammatory co-morbidities and time-of-day therapeutics.

Polyomaviruses BK and JC DNA infection in peripheral blood cells from blood donors

ObjectivesTo investigate the prevalence of human polyomavirus (BK and JC viruses) infection in peripheral blood mononuclear cells of healthy blood donors. MethodsThe study included 250 healthy blood donors. Five-milliliter blood was drawn into sterile EDTA tubes and PBMCs were isolated from whole blood. The isolated PBMCs were counted and stored at −70°C for future investigation. DNA was extracted and subjected to simple, sensitive and specific semi-nested PCR as well as QPCR using both general and specific primers for different assays. ResultsOf 250 blood samples, 66 (26.4%) were positive for BKV DNA (146–34,514 copies/106 cells). JC DNA was found in 45 (18%) blood samples (65–21,250 copies/106 cells). Co-infection with these viruses were found in 11 (4.4%) out of 250 blood samples. DiscussionOur study provides important data on polyomavirus infection in peripheral blood mononuclear leukocytes in immunocompetent individuals. These data indicate significant differences between the prevalence of BKV and JCV infection in healthy blood donors. The prevalence of BK and JC virus infection is higher in the age range 30–39 years compared to other age ranges.

Development of secondary mitochondrial dysfunction of mononuclear blood leukocytes in patients with chronic obstructive pulmonary disease and chronic bronchitis

Aim. To study the indicators of energy metabolism and oxidative stress in mononuclear leukocytes of peripheral blood and to assess the possibility of mitochondrial dysfunction development in chronic obstructive pulmonary disease and chronic bronchitis.
 Methods. The study included 50 patients aged 40 to 75 years with chronic obstructive pulmonary disease (COPD) or chronic bronchitis. The first group included 13 patients with chronic bronchitis. In accordance with the GOLD spirometric classification, the second and third groups included patients with COPD of moderate severity (COPD 2) (n=17) and severe COPD (COPD 3) (n=20) respectively. In the isolated mononuclear leukocytes, the activity of superoxide dismutase (SOD), succinate dehydrogenase (SDH) and concentration of succinate were determined, a complex evaluation of oxidative modification of proteins was performed.
 Results. Patients with chronic bronchitis compared to patients with COPD 2 and COPD 3 were found to have in mononuclear leukocytes higher activity of SOD by 3.38 times (p=0.0025) and 3.15 times (p=0.0058), higher activity of SDH by 4.55 times (p=0.0281) and 2.5 times (p=0.0263) and higher succinate concentration by 2.05 (p=0.0133) and 1.89 (p=0.005) times respectively. The level of spontaneously oxidized modified proteins in the group of patients with chronic bronchitis decreased by 2.45 (p=0.0176) and 2.94 (p=0.0168) times compared to the patients of groups 2 and 3, respectively There was a decrease in the reserve-adaptive potential of oxidative modification of proteins in COPD in the form of an increase of the ratio of spontaneously oxidized-modified proteins to metal-induced oxidized proteins by 1.58 times (p=0.0301) between groups 1 and 2, and by 1.44 times between groups 2 and 3 (p=0.0446).
 Conclusion. In mononuclear leukocytes of COPD patients, secondary mitochondrial dysfunction is observed accompanied by significant oxidative damage of lymphocytes and monocytes. Patients with severe COPD compared to patients with COPD of moderate severity have less reserve-adaptive potential for the oxidative modification of mononuclear leukocyte proteins, which probably reflects a more severe course of the disease.

Cytoprotective and Anti-genotoxic Effects of Xanthone Derivatives from Garcinia mangostana Against H2O2 Induced PBMC Cell and Blood Leukocytes Damage of Normal and Type 2 Diabetes Volunteers

Hyperglycemia is well-known for inducing cellular oxidative damage in type II diabetes (T2D) patients. This research addressed the cytoprotective and anti-genotoxic effect of xanthone derivatives from Garcinia mangostana against hydrogen peroxide (H2O2)-induced human peripheral blood mononuclear cell (PBMC) and blood leukocytes damage of the normal and T2D volunteers. The cytoprotective effects of an aqueous extract of xanthone (100 and 200 µg/mL) was assessed on cell viability and free radical scavenging activity using the trypan blue exclusion method on PBMC cells. Malondialdehyde (MDA) levels and lactate dehydrogenase (LDH) activity were measured as cellular oxidative damage markers and estimated from culture medium of PBMCs of normal and T2D volunteers. The anti-genotoxicity was assessed as the protective effect of xanthone against H2O2-induce DNA damage of blood leukocytes of the normal volunteers following comet assay technique. Xanthone and Gallic acid (control) concentrations 100, 200 and 100 µg/mL significantly (P < 0.05) protected from H2O2 (20 mM)-induced oxidative damage of PBMCs. It was confirmed by increased cell viability and free radical scavenging activity coupled with the decreased MDA and LDH levels in cell culture medium compared to H2O2 (20 mM)-treated group. In H2O2 (40 mM)-induced blood leukocytes of normal volunteers, different concentration xanthone (50 - 500 µg/mL) significantly (P < 0.05) improved the anti-genotoxicity effect compared to negative/positive control group by lowering comet formation. Xanthone treatments on PBMCs and blood leukocytes of the normal and T2D volunteers could attenuate the H2O2-induced cellular oxidative damage and cell death via exhibiting antioxidant and free radical scavenging activities.

Do Progestin-Only Contraceptives Contribute to the Risk of Developing Depression as Implied by Beta-Arrestin 1 Levels in Leukocytes? A Pilot Study.

We reported previously that reduction in beta-arrestin 1 (β-AR 1) protein levels in peripheral blood mononuclear leukocytes (PBMC) significantly correlated with the severity of depressive symptoms in reproductive women. In this pilot study, we used β-AR 1 protein levels in PBMC as a marker for developing depressive symptoms and the Hamilton Depression Rating Scale (HAM-D) scores to assess potential mood-related side effects of oral contraceptive use for routine birth control among women. We evaluated 29 women in this study. We enrolled the participants in three groups: Estrogen-progestin combination-oral contraceptives (COC, n = 10), progestin-only contraceptives (POC, n = 12), and non-hormonal or no contraceptives (NC, n = 7). We determined the β-AR 1 protein levels in PBMCs by enzyme-linked immunosorbent assay (ELISA). We found that women in the POC group had significantly higher HAM-D scores compared to those in the COC (p < 0.0004) and NC (p < 0.004). The levels of β-AR 1 protein were significantly attenuated in women in the POC group compared to women in the NC group (p = 0.03). Our findings suggest that the use of POC is a potential risk factor for developing depressive symptoms.

C-Reactive Protein Stimulates Nicotinic Acetylcholine Receptors to Control ATP-Mediated Monocytic Inflammasome Activation.

Blood levels of the acute phase reactant C-reactive protein (CRP) are frequently measured as a clinical marker for inflammation, but the biological functions of CRP are still controversial. CRP is a phosphocholine (PC)-binding pentraxin, mainly produced in the liver in response to elevated levels of interleukin-1β (IL-1β) and of the IL-1β-dependent cytokine IL-6. While both cytokines play important roles in host defense, excessive systemic IL-1β levels can cause life-threatening diseases such as trauma-associated systemic inflammation. We hypothesized that CRP acts as a negative feedback regulator of monocytic IL-1β maturation and secretion. Here, we demonstrate that CRP, in association with PC, efficiently reduces ATP-induced inflammasome activation and IL-1β release from human peripheral blood mononuclear leukocytes and monocytic U937 cells. Effective concentrations are in the range of marginally pathologic CRP levels (IC50 = 4.9 µg/ml). CRP elicits metabotropic functions at nicotinic acetylcholine (ACh) receptors (nAChRs) containing subunits α7, α9, and α10 and suppresses the function of ATP-sensitive P2X7 receptors in monocytic cells. Of note, CRP does not induce ion currents at conventional nAChRs, suggesting that CRP is a potent nicotinic agonist controlling innate immunity without entailing the risk of adverse effects in the nervous system. In a prospective study on multiple trauma patients, IL-1β plasma concentrations negatively correlated with preceding CRP levels, whereas inflammasome-independent cytokines IL-6, IL-18, and TNF-α positively correlated. In conclusion, PC-laden CRP is an unconventional nicotinic agonist that potently inhibits ATP-induced inflammasome activation and might protect against trauma-associated sterile inflammation.

Glucocorticoid receptor isoform expression in peripheral blood mononuclear leukocytes of patients with chronic rhinosinusitis.

In several inflammatory disorders, altered peripheral blood mononuclear leukocyte (PBML) glucocorticoid (GC) receptor isoform expression has been associated with GC resistance and disease severity. However, it is unclear if PBML GC receptor isoforms are expressed differentially and are associated with worsened disease severity in chronic rhinosinusitis (CRS). PBMLs were isolated from control (n = 8), CRS without nasal polyps (CRSsNP) (n = 8), atopic CRS with nasal polyps (CRSwNP) (n = 8), non-atopic CRSwNP (n = 8), and allergic fungal rhinosinusitis (AFRS) (n = 8) patients. Demographics, atopic status, asthmatic status, 22-item Sino-Nasal Outcome Test (SNOT-22) scores, Lund-Kennedy nasal endoscopy scores, Lund-Mackay sinus computed tomography (CT) scores, Kennedy Osteitis scores, and GC utilization 6 months postoperatively were collected. Intracellular immunostaining was then performed for functional GC receptor α (GCRα) and nonfunctional GC receptor β (GCRβ), followed by flow cytometry analysis of geometric mean fluorescent intensity (MFI) and the percentage of cells expressing each GC receptor isoform. Compared to controls, each CRS subtype had decreased PBML GCRα and GCRα:GCRβ MFI expression, but no difference in GCRβ expression. Decreasing PBML GCRα in AFRS was associated with increasing Lund-Mackay sinus CT scores (r = -0.880, p =0.004). No significant associations were found between GC receptor isoform expression and other clinical measures. CRS patients have reduced functional PBML GCRα expression and decreased GCRα:GCRβ compared to controls. Reductions in GCRα in AFRS are associated with worsening Lund-Mackay sinus CT scores. The clinical implications of decreased functional GC receptor expression merits further investigation.

Skin neurogenic inflammation.

The epidermis closely interacts with nerve endings, and both epidermis and nerves produce substances for mutual sustenance. Neuropeptides, like substance P (SP) and calcitonin gene-related protein (CGRP), are produced by sensory nerves in the dermis; they induce mast cells to release vasoactive amines that facilitate infiltration of neutrophils and T cells. Some receptors are more important than others in the generation of itch. The Mas-related G protein-coupled receptors (Mrgpr) family as well as transient receptor potential ankyrin 1 (TRPA1) and protease activated receptor 2(Par2) have important roles in itch and inflammation. The activation of MrgprX1 degranulates mast cells to communicate with sensory nerve and cutaneous cells for developing neurogenic inflammation. Mrgprs and transient receptor potential vanilloid 4 (TRPV4) are crucial for the generation of skin diseases like rosacea, while SP, CGRP, somatostatin, β-endorphin, vasoactive intestinal peptide (VIP), and pituitary adenylate cyclase-activating polypeptide (PACAP) can modulate the immune system during psoriasis development. The increased level of SP, in atopic dermatitis, induces the release of interferon (IFN)-γ, interleukin (IL)-4, tumor necrosis factor (TNF)-α, and IL-10 from the peripheral blood mononuclear leukocytes. We are finally starting to understand the intricate connections between the skin neurons and resident skin cells and how their interaction can be key to controlling inflammation and from there the pathogenesis of diseases like atopic dermatitis, psoriasis, and rosacea.

A pilot study on the effects of hormonal contraceptives on beta‐arrestin1 levels in peripheral blood mononuclear leukocytes

OBJECTIVEWomen are twice as likely as likely as men to develop depression during reproductive years. Use of hormonal contraceptives, during reproductive years, may be causal with development of depression in a subgroup of women because observational studies indicate an association between use of progestin (Depo‐Provera) and development of depressive symptoms. Previously we have reported reduced levels of beta arrestin 1(β‐AR‐1) during premenstrual depressive disorder, may be a potential biomarker of depression. In this study we compared the β‐AR‐1 protein levels and the Hamilton Depression Rating Scores (HDRS) as well, in women on non‐hormonal contraceptives (control), combine‐only contraceptives (COC), and progestin‐only contraceptives (POC).METHODSStudy participants (n=29) were non‐pregnant women between 18–42 years of age, not taking any antidepressants/therapy and at the luteal phase of menstruation. The participants were evaluated with Neuropsychiatric Interview (DSMIV‐TR). The severity of depression were determined by the HDRS. The protein levels of beta‐arrestin1 in the in peripheral blood mononuclear leukocytes (PBMC) were determined by ELISA.RESULTSThe magnitude of the different parameters for Axis 1 mental disorders were significantly higher and beta arrestin1 protein levels in PBMC were significantly lower in women currently using progestin‐only contraceptives as compared to the control.CONCLUSIONThese findings indicate the possibility of reduced levels of β‐AR‐1 and associated symptoms of depression in a subgroup of women may results from the use of POC.Support or Funding InformationClinical Research Education and Career Development in Minority R25RR17577 (NCRR/NIMHD) and U54 MD007593 (NIMHD) and U54 RR026140 (NCRR) at NIH.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Targeted bisulfite sequencing identified a panel of DNA methylation-based biomarkers for esophageal squamous cell carcinoma (ESCC)

BackgroundDNA methylation has been implicated as a promising biomarker for precise cancer diagnosis. However, limited DNA methylation-based biomarkers have been described in esophageal squamous cell carcinoma (ESCC).MethodsA high-throughput DNA methylation dataset (100 samples) of ESCC from The Cancer Genome Atlas (TCGA) project was analyzed and validated along with another independent dataset (12 samples) from the Gene Expression Omnibus (GEO) database. The methylation status of peripheral blood mononuclear cells and peripheral blood leukocytes from healthy controls was also utilized for biomarker selection. The candidate CpG sites as well as their adjacent regions were further validated in 94 pairs of ESCC tumor and adjacent normal tissues from the Chinese Han population using the targeted bisulfite sequencing method. Logistic regression and several machine learning methods were applied for evaluation of the diagnostic ability of our panel.ResultsIn the discovery stage, five hyper-methylated CpG sites were selected as candidate biomarkers for further analysis as shown below: cg15830431, P = 2.20 × 10−4; cg19396867, P = 3.60 × 10−4; cg20655070, P = 3.60 × 10−4; cg26671652, P = 5.77 × 10−4; and cg27062795, P = 3.60 × 10−4. In the validation stage, the methylation status of both the five CpG sites and their adjacent genomic regions were tested. The diagnostic model based on the combination of these five genomic regions yielded a robust performance (sensitivity = 0.75, specificity = 0.88, AUC = 0.85). Eight statistical models along with five-fold cross-validation were further applied, in which the SVM model reached the best accuracy in both training and test dataset (accuracy = 0.82 and 0.80, respectively). In addition, subgroup analyses revealed a significant difference in diagnostic performance between the alcohol use and non-alcohol use subgroups.ConclusionsMethylation profiles of the five genomic regions covering cg15830431 (STK3), cg19396867, cg20655070, cg26671652 (ZNF418), and cg27062795 (ZNF542) can be used for effective methylation-based testing for ESCC diagnosis.

Reduced activation and proliferation of human lymphocytes exposed to respiratory syncytial virus compared to cells exposed to influenza virus.

Both respiratory syncytial virus (RSV) and influenza A virus (IAV) may infect human peripheral blood mononuclear leukocytes (PBMC) during the immune response to viral challenge as the cells are recruited to the respiratory tract. The current studies demonstrated differences in PBMC responses to the two viruses very early after exposure, including reduced fos protein and CD69 expression and IL-2 production by RSV-exposed T lymphocytes. Exposure to RSV resulted in reduced lymphocyte proliferation despite evidence of a virus-specific T lymphocyte frequency equivalent to that for influenza virus. Reduced RSV-induced proliferation was not due to apoptosis, which was itself reduced relative to that of influenza virus-exposed T lymphocytes. The data indicate that differential immune responses to RSV and influenza virus are determined early after exposure of human PBMC and support the concept that the anamnestic immune response that might prevent clinically evident reinfection is attenuated very soon after exposure to RSV. Thus, candidate RSV vaccines should be expected to reduce but not prevent clinical illness upon subsequent infection by RSV. Furthermore, effective therapeutic agents for RSV are likely to be needed, especially for high-risk populations, even after vaccine development.

Isolation and characterization of monocyte/macrophage from peripheral blood of half smooth tongue sole (Cynoglossus semilaevis)

In the present study, the peripheral blood cells of half smooth tongue sole (Cynoglossus semilaevis) were examined by blood smear under the light microscopy. The proportion of main types of blood cells are as following: erythrocyte occupied the majority (92.3%), followed by thrombocyte (4.15%), granulocyte (1.7%), lymphocyte (1.5%) and monocyte (0.3%), respectively. Meanwhile, the isolation method of monocytes was established, by density gradient centrifugation to isolate mononuclear leukocytes of peripheral blood. In primary culture, the monocytes were adhered to the bottom of the flask without feeder cells and separated easily with suspended leukocytes in the medium in 3 h. After suspended leukocytes were removed, the monocytes multiplied rapidly with the two doubly during the 24 h, then the cells proliferated and kept stable until 48 h. When co-cultured with suspended leukocytes after three days, the monocytes could derive to typical macrophages, of which the size enlarged significantly and showed various forms such as like fried eggs, and giant irregular shape with pseudopod because cells fusion or deformation occurred until macrophages died in about two weeks. Monocytes showed strong respiratory burst activity after treated with Phorbol ester PMA and challenged by bacteria respectively. In addition, macrophage of half smooth tongue sole had typical macrophage features such as phagocytic capability, positive esterase activity, and the considerable expression of M-CSFR, MHC-II, IL-6, IL-10, TNF and arginase genes. That arginase expression in macrophages (3d and 5d after differentiation) was upregulated fluctuant suggest that the cultivation was mixture of alternatively activated type macrophage (M2) in the majority while the classically activated type (M1) win the minority. Furthermore, MHC-Ⅱ, M-CSFR and IL-6 were significantly induced following LPS challenge. Collectively, the present study will be useful for the study on half smooth tongue sole immune systems and immune function.

Fibronectin connecting segment-1 peptide inhibits pathogenic leukocyte trafficking and inflammatory demyelination in experimental models of chronic inflammatory demyelinating polyradiculoneuropathy

The molecular determinants of pathogenic leukocyte migration across the blood-nerve barrier (BNB) in chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) are unknown. Specific disease modifying therapies for CIDP are also lacking. Fibronectin connecting segment-1 (FNCS1), an alternatively spliced fibronectin variant expressed by microvascular endothelial cells at sites of inflammation in vitro and in situ, is a counterligand for leukocyte α4 integrin (also known as CD49d) implicated in pathogenic leukocyte trafficking in multiple sclerosis and inflammatory bowel disease. We sought to determine the role of FNCS1 in CIDP patient leukocyte trafficking across the BNB in vitro and in severe chronic demyelinating neuritis in vivo using a representative spontaneous murine CIDP model. Peripheral blood mononuclear leukocytes from 7 untreated CIDP patients were independently infused into a cytokine-treated, flow-dependent in vitro BNB model system. Time-lapse digital video microscopy was performed to visualize and quantify leukocyte trafficking, comparing FNCS1 peptide blockade to relevant controls. Fifty 24-week old female B7-2 deficient non-obese diabetic mice with spontaneous autoimmune peripheral polyneuropathy (SAPP) were treated daily with 2mg/kg FNCS1 peptide for 5days via intraperitoneal injection with appropriate controls. Neurobehavioral measures of disease severity, motor nerve electrophysiology assessments and histopathological quantification of inflammation and morphometric assessment of demyelination were performed to determine in vivo efficacy. The biological relevance of FNCS1 and CD49d in CIDP was evaluated by immunohistochemical detection in affected patient sural nerve biopsies. 25μM FNCS1 peptide maximally inhibited CIDP leukocyte trafficking at the human BNB in vitro. FNCS1 peptide treatment resulted in significant improvements in disease severity, motor electrophysiological parameters of demyelination and histological measures of inflammatory demyelination. Microvessels demonstrating FNCS1 expression and CD49d+ leukocytes were seen within the endoneurium of patient nerve biopsies. Taken together, these results imply a role for FNCS1 in pathogenic leukocyte trafficking in CIDP, providing a potential target for therapeutic modulation.

The Protective Effect of Naringenin-Oxime on Cisplatin-Induced Toxicity in Rats.

The aim of this study is to examine the protective effect of naringenin-oxime (NOX) on cisplatin-induced major organ toxicity and DNA damage in rats. Thirty-five male Wistar albino rats were equally split into five groups as follows: control (i.p., 0.1 ml of saline), Cis administration (i.p., 7 mg/kg b.w.), NOX treatment (i.p., 20 mg/kg b.w., daily for ten days), Cis + NOX20, and Cis + NOX40 combination (i.p., 20 and 40 mg/kg b.w., daily for ten days). Serum and peripheral blood mononuclear leukocytes (PBMC) were obtained from blood. Malondialdehyde, glutathione, total antioxidant and oxidant status, and catalase were measured in serum, liver, and kidney, and oxidative stress index was calculated. In parallel, paraoxonase and arylesterase activities were tested in liver and serum. We used 8-OHdOG as a marker for DNA damage in serum via ELISA and in PMBC via comet assay. Treatment with Cis elevated the levels of serum biochemical parameters, oxidative stress, and DNA damage. Pretreatments of NOX restored biochemical and oxidative stress parameters in serum, renal, and liver tissues (p < 0.01) and reduced 8-OHdG level, a finding further supported by comet assay in PBMC. Observations of the present study support the fact that treatment with NOX prevents Cis-induced hepatotoxicity, nephrotoxicity, and genotoxicity by restoring antioxidant system.

Cellular and molecular screening of connective tissue dysplasia in adolescent athletes (pilot study)

The purpose of the study is to evaluate the cellular and molecular parameters of bone remodeling in the blood as potential markers of undifferentiated forms of connective tissue dysplasia Materials and methods. The structural and functional status of cellular elements of in vitro culturing of mononuclear leukocytes of peripheral blood in adolescent athletes connected with phenotypic manifestations of undifferentiated connective tissue dysplasia (UCTD) were investigated. 25 pupils of sport schools from 10–14 years old (main disciplines: figure skating, gymnastics, athletics) were examined with the help of express analysis. The average age of the examined adolescents was (12,0 ± 1,7) years. Clinical examination of adolescents allowed ranking the UCTD signs on a scale of 4–11,5 points. Results. A comparison of questionnaire survey results and an evaluation of bone remodeling distant markers allowed the revelation of 2 groups in the distribution of adolescent athletes: those with minimal signs of UCTD (scores lesser than 7 points – 10 pupils), and those with expressed UCTD phenotype (scores are equal or more than 7 points –15 pupils). Significant statistical decrease in the content of collagen type I degradation products (CrossLaps) (by 80%) and ionized calcium (by 5%) has been determined in the peripheral blood of adolescent athletes with expressed UCTD phenotype. In conditions of short-term 72-h cultivation of mononuclear leukocytes in the presence of a 3D matrix imitating the properties of the mineral substance of the regenerating bone tissue, morphofunctional features of cellular reaction in adolescent athletes with clinical manifestations of UCTD, as well the heterogeneity of the cell population associated with the appearance of cells with an osteoblast-like phenotype in the blood have been revealed. The results of investigation propose the use of distant cellular and molecular parameters of bone remodeling to screen the mechanisms and dynamics of dysplastic disorders in adolescent athletes.

EFFECTIVENESS OF APPLICATION OF COMBINED THERAPY OF EARLY ERYTHEMA STAGE OF LYME DISEASE TAKING INTO CONSIDERATION IMMUNOGENESIS FEATURES

Study innate and adaptive immunity in patients with migrating erythema, clinical effectiveness ofcombined therapy using Immunovac vaccine and dynamics of immunologic parame- ters as a result of the therapy. 37 adult patients with migrating erythema were examined. The patients were divided into 2 groups: 1st gr. (14 individuals) - Immunovac by intranasal-subcutaneous method against the background of basic therapy; 2nd gr. (23 individuals) - 200 mg/day doxycycline therapy for 21 days. Phagocytic activity of blood neutrophils; TLRs expression on mononuclear leukocytes of peripheral blood (PBML) and skin cells in foci by flow cytometry with mAT against TLR2, 3, 4, 5, 6, 7, 8, 9 using flow cytometer FC-500; subpopulation composition of peripheral blood lymphocytes; levels of pro-, anti-inflammation and regulatory cytokines in blood sera by EIA method; IgG, 1gM, IgA in blood sera were studied in patients before treatment and 1 month after therapy. A high level of TLR2, 4, 7, 8 on skin cells in foci, TLR2, 4 - on blood cells; low content of CD95+ and CD25+, high-level of serum IL-lb, IL-2 and IL-4, an increase of general IgE level was detected in patients'with migrating erythema. Immunovac facilitated an increase of CD95+ and CD25+, IFN-y synthesis, reduced the level of general IgE in a more pronounced way than basic therapy. Inclusion of Immunovac into therapy of patients With migrating erythema facilitates increase of clinical effectiveness and correlates with correction of immunologic disorders.

Concurrent targeting Akt and sphingosine kinase 1 by A-674563 in acute myeloid leukemia cells

Akt signaling plays a pivotal role in acute myeloid leukemia (AML) development and progression. In the present study, we evaluated the potential anti-AML activity by a novel Akt kinase inhibitor A-674563. Our results showed that A-674563 dose-dependently inhibited survival and proliferation of U937 AML cells and six lines of human AML progenitor cells, yet sparing human peripheral blood mononuclear leukocytes (PBMCs). A-674563 activated caspase-3/9 and apoptosis in the AML cells. Reversely, the pan-caspase inhibitor z-VAD-CHO dramatically alleviated A-674563-induced AML cell apoptosis and cytotoxicity. For the molecular study, we showed that A-674563 blocked Akt activation in U937 cells and human AML progenitor cells. Further, A-674563 decreased sphingosine kinase 1 (SphK1) activity in above AML cells to deplete pro-survival sphingosine-1-phosphate (S1P) and boost pro-apoptotic ceramide production. Such an effect on SphK1 signaling by A-674563 appeared independent of Akt blockage. Significantly, K6PC-5, a novel SphK1 activator, or supplement with S1P attenuated A-674563-induced ceramide production, and subsequent U937 cell death and apoptosis. Importantly, intraperitoneal injection of A-674563 at well-tolerated doses suppressed U937 leukemic xenograft tumor growth in nude mice, whiling significantly improving the animal survival. The results of the current study demonstrate that A-674563 exerts potent anti-leukemic activity in vitro and in vivo, possibly via concurrent targeting Akt and SphK1 signalings.

Preclinical evaluation of WYE-687, a mTOR kinase inhibitor, as a potential anti-acute myeloid leukemia agent

Mammalian target of rapamycin (mTOR) as a potential drug target for treatment of acute myeloid leukemia (AML). Here, we investigated the potential anti-leukemic activity by WYE-687, a potent mTOR kinase inhibitor. We demonstrated that WYE-687 potently inhibited survival and proliferation of established (HL-60, U937, AML-193 and THP-1 lines) and human AML progenitor cells. Yet, same WYE-687 treatment was non-cytotoxic to the primary peripheral blood mononuclear leukocytes (PBMCs) isolated from healthy donors. WYE-687 induced caspase-dependent apoptotic death in above AML cells/progenitor cells. On the other hand, the pan-caspase inhibitor (Z-VAD-FMK), the caspase-3 specific inhibitor (Z-DEVD-FMK) or the caspase-9 specific inhibitor (z-LEHD-fmk) attenuated WYE-687-induced cytotoxicity. At the molecular level, WYE-687 concurrently inhibited activation of mTORC1 (p70S6K1 and S6 phosphorylations) and mTORC2 (AKT Ser-473 and FoxO1/3a phosphorylations), whiling downregulating mTORC1/2-regulated genes (Bcl-xL and hypoxia-inducible factor 1/2α) in both HL-60/U937 cells and human AML progenitor cells. In vivo, oral administration of WYE-687 potently inhibited U937 leukemic xenograft tumor growth in severe combined immunodeficient (SCID) mice, without causing significant toxicities. In summary, our results demonstrate that targeting mTORC1/2 by WYE-687 leads to potent antitumor activity in preclinical models of AML.