Maja Milojkovic

Frequency of the C1236T, G2677T/A and C3435T MDR1 gene polymorphisms in the Serbian population.

The multi-drug resistance 1 (MDR1) gene encodes for a P-glycoprotein (PGP), which acts as a gate-keeper against various kinds of xenobiotics. Several single nucleotide polymorphisms (SNPs) in the MDR1 gene that may influence PGP level and function have been identified. The aim of this study was to simultaneously analyze the three most important MDR1 SNPs, C3435T, G2677T/A and C1236T, in the Serbian population and to compare the results with those published for other ethnic groups. A group of 158 unrelated, healthy subjects was included in the present study. For determination of MDR1 SNPs, a multiplexed mutagenically separated PCR was performed. The genotype frequency of the analyzed MDR1 SNPs was as follows: 3435 nt - 0.19 (CC), 0.54 (CT) and 0.27 (TT); 2677 nt - 0.26 (GG), 0.52 (GT), 0.15 (TT), 0.03 (GA) and 0.064 (TA), and 1236 nt - 0.23 (CC), 0.61 (CT) and 0.16 (TT). Our results for the Serbian population could be relevant for further investigation of drugs that are substrates of PGPand for studies of interethnic diversity in MDR1 polymorphism frequency.

Association of serum bilirubin and uric acid levels changes during neuroinflammation in patients with initial and relapsed demyelination attacks

In order to examine the endogenous antioxidants values in the earliest phase of demyelination, we have determined bilirubin and uric acid (UA) serum values in the patients with clinically isolated syndrome (CIS) and relapsing remitting multiple sclerosis (RRMS), regarding their clinical disability, measured by Extended Disability Status Scale (EDSS), Magnetic Resonance Imaging (MRI), disease duration, gender and other parameters. The bilirubin and UA levels were lower in CIS and RRMS patients than in control group, whether male or female (p < 0.05). The bilirubin and UA levels were decreased in RRMS compared to CIS patients (p < 0.05). Regarding EDSS, MRI and disease duration, obtained values of bilirubin and UA were higher in both study groups in patients with lower EDSS, lower MRI lesion number and shorter disease duration (p < 0.05). The greatest significance in decreased bilirubin and UA levels was observed in female compared to male patients, in both study groups (p < 0.05). The results suggest negative linear correlation between bilirubin and UA levels and disease duration, EDSS and MRI in CIS (p < 0.01), with the same correlation between bilirubin and UA levels and disease duration in RRMS patients (p < 0.01). There was also significant correlation between bilirubin level and MRI findings and UA levels and EDSS in RRMS patients (p < 0.01). The obtained results point to the importance of endogenous antioxidants in the outbreak and course of neuroinflammation. This could be favorable for the new pathogenetically conditioned neuroinflammatory therapy concepts which do not initially rely only on immunomodulatory, but also on the antioxidative effects.

Crosstalk of Various Biomarkers That Might Provide Prompt Identification of Acute or Chronic Cardiorenal Syndromes

Introduction: Pathophysiological interaction between the heart and kidneys represents the basis for clinical entities called cardiorenal syndromes. The purpose of the study was to assess the relations between acute and chronic cardiorenal syndromes and biomarkers [advanced oxidation protein products, brain natriuretic peptide, malondialdehyde, xanthine oxidoreductase (XOD), xanthine oxidase, xanthine dehydrogenase, interleukin 8, cystatin C, plasminogen activator inhibitor-1, high-sensitive troponin T, C-reactive protein and glomerular filtration rate, measured by the Modification of Diet in Renal Disease (MDRD) formula], to hypothesize biomarkers that might provide a prompt identification of acute or chronic cardiorenal syndromes, and to distinguish acute versus chronic types of these syndromes. Methods: A total of 114 participants were enrolled in this study, i.e. 79 patients divided into subgroups of acute and chronic cardiorenal syndromes and 35 volunteers. Results: Nonadjusted odds ratio (OR) showed that there was a significant risk for acute cardiorenal syndrome with increased XOD activity (p = 0.037), elevated cystatin C concentration (p = 0.038) and MDRD (p = 0.028). Multivariable adjusted OR, on the other hand, revealed that only glomerular filtration rate measured by the MDRD formula had a significance for acute cardiorenal syndrome (p = 0.046). Nonadjusted OR showed a significant risk for chronic cardiorenal syndrome only in elderly (p = 0.002). Multivariable adjusted OR exhibited that age was the only risk factor for chronic cardiorenal syndrome (p = 0.012). Conclusion: Cystatin C, glomerular filtration rate measured by the MDRD equation and XOD were independent risk factors for acute cardiorenal syndrome, while age remained an independent risk factor for chronic cardiorenal syndrome. When comparing ORs of evaluated parameters, the highest significance for acute cardiorenal syndrome was plasma concentration of cystatin C.

LIPID PEROXIDATION AND OXIDATIVE PROTEIN PRODUCTS IN CHILDREN WITH EPISODIC FEVER OF UNKNOWN ORIGIN / LIPIDNA PEROKSIDACIJA I OKSIDATIVNI PROTEINSKI PRODUKTI KOD DECE SA EPIZODINOM GROZNICOM NEPOZNATOG UZROKA

Summary Background: Episodic fever syndromes are commonly seen in pediatric practice. Episodic fever of unknown origin (FUO) lasts for a few days or weeks and is followed by a fever-free period with a sense of well-being. In this condition, activated neutrophils and monocytes intensively generate reactive oxidative species that may further damage various mole- cules. The aim of the study was to evaluate oxidative stress biomarkers, lipid peroxidation in erythrocytes and plasma, and advanced oxidation protein products (AOPP) in children with episodic FUO. Methods: The study enrolled 25 children with episodic FUO in afebrile phase and 25 healthy children as controls. Lipid peroxidation was evaluated by measuring malondialdehyde (MDA) production with the thiobarbituric-acid-reactive sub- stances (TBARS) assay in erythrocytes and plasma. Oxidative modification of proteins was measured spectrophotometri- cally by the determination of AOPP in plasma. Results: Mean duration of episodic fevers was 3.96±2.8 years. Erythrocyte MDA levels were higher in children with FUO than in controls (86.26± 10.75 vs. 78.0±3.21 nmol/g hemoglobin), although not significantly (p=0.202). The MDA plasma concentrations were similar (2.42±0.35 vs. 2.41 ±0.39 (xmol/L) between the groups (p=0.732). Unexpectedly, levels of AOPP were significantly lower in chil- dren with FUO than in healthy controls (18.8±5.04 vs. 25.1 ±3.35 nmol/L, p=0.047). Conclusions: Episodic fevers of unknown origin with an aver- age duration of 3.96±2.8 years do not cause significant oxidative modifications of lipids and proteins in children. Kratak sadržaj Uvod: Sindromi epizodične ili rekurentne groznice često se sreću u pedijatrijskoj praksi. Epizodična groznica nepoznatog uzroka (FUO) traje nekoliko dana do nekoliko nedelja, nakon čega sledi miran period bez povišene temperature uz osećaj potpunog zdravlja. U ovim stanjima, aktivirani neutrofili i monociti intenzivno produkuju reaktivne kiseonične vrste koje naknadno mogu oštetiti različite molekule. Cilj našeg rada bio je oceniti biomarkere oksidativnog stresa, odnosno lipidnu peroksidaciju u eritrocitima i plazmi, kao i uznapredovale oksidativne proteinske produkte (AOPP) kod dece sa epizo- dičnom FUO. Metode: U istraživanje je uključeno 25 dece sa epizodičnom groznicom u afebrilnoj fazi i 25 zdrave dece. Lipidna peroksi- dacija je utvrđena merenjem produkcije malondialdehida (MDA) korišćenjem testa tiobarbiturnih reagujućih supstanci (TBARS) u eritrocitima i plazmi. Oksidativna modifikacija proteina je određivana spektrofotometrijski, merenjem AOPP-a u plazmi. Rezultati: Srednje vreme trajanja epizodičnih groznica bilo je 3,96±2,8 godina. Vrednosti MDA u eritrocitima su bile vise kod dece sa epizodičnom FUO nego kod zdrave dece (86,26 ±0,75 vs. 78,0±3,21 nmol/g hemoglobina), iako ne sta- tistički značajno (p=0,202). Koncentracije MDA u plazmi su bile slične kod ove dve grupe dece (2,42±0,35 vs. 2,41 ± 0,39(jmol/L, p=0,732). Neočekivano, nivoi AOPP-a su zna- čajno bili manji kod dece sa FUO nego kod zdravih kontrol- nih subjekata (18,8±5,04 vs. 25,1 ±3,35 (jmol/L, p=0,047). Zaključak: Epizodične groznice nepoznatog uzroka u trajanju od 3,96 ±2,8 godina ne izazivaju značajnu oksidativnu mo- difikaciju lipida i proteina kod dece.

THE CELL-FREE DNA DETECTION AND ANALYSIS AS THE NEW NON-INVASIVE PRENATAL DIAGNOSTICS OPTION

Screening procedures for chromosomal abnormalities in fetuses are a standard of care for pregnant women. Ultrasound and maternal serum analysis are traditional prenatal screening methods with detection rates between 75%-95%, and considerable false-negative and false-positive results. Also, both require follow up by invasive diagnostic tests in screen-positive cases, mostly amniocentesis and chorionic villi sampling, which are associated with notable risk of pregnancy loss. One of the innovative non-invasive prenatal testing (NIPT) options is the analysis of cell-free DNA (cfDNA) in plasma, which is detected in maternal circulation in a relatively high concentration. Commercial tests for cfDNA in maternal blood have recently become available. Cell-free DNA detection tests do not separate fetal from maternal DNA but use full cfDNA complement and analyze difference in total amount of sequenced DNA fragments, with the help of sophisticated data analysis software. It seems that cfDNA technology testing is highly accurate and has a very high sensitivity, so the difference compared to routine serum sample screening shows its significant superiority. However, cfDNA positive results still need confirmation by the invasive testing. The cell-free DNA analysis aims to become the first choice NIPT option due to its safety and high accuracy rate. The final goal is to develop the reliable method that could eventually replace invasive prenatal testing procedures.

Mechanisms of plasminogen activator inhibitor 1 action in stromal remodeling and related diseases

Plasminogen activator inhibitor type 1 (PAI-1) is the main physiologic inhibitor of fibrinolysis. However, it is also involved in many physiological processes such as extracellular matrix (ECM) proteolysis and remodeling, cell adhesion, motility, and apoptosis, angiogenesis, etc. The aim of the study was to summarize current knowledge and gain insights into the mechanisms of PAI-1 action in the processes of stromal remodeling and diseases with considerable matrix pathologies (atherosclerosis, tissue fibrosis, cancer metastasis, pregnancy related complications, etc). As a component of an early cellular response to injury, PAI-1 reacts with membrane surface proteins and participates in the initiation of intracellular signaling, specifically cytoskeletal reorganization and motility. Complexity of ECM homeostasis resides in varying relation of the plasminogen system components and other matrix constituents. Inflammatory mediators (transforming growth factor-β and interferon-γ) and hormones (angiotensin II) are in the close interdependent relation with PAI-1. Also, special attention is devoted to the role of increased PAI-1 concentrations due to the common 4G/5G polymorphism. Some of the novel mechanisms of ECM modification consider PAI-1 dependent stabilization of urokinase mediated cell adhesion, control of the vascular endothelial cadherin trafficking and interaction with endothelial cells proteasome, its relation to matrix metalloproteinase 2 and osteopontin, and oxidative inhibition by myeloperoxidase. Targeting and/or alteration of PAI-1 functions might bring benefit to the future therapeutic approaches in diseases where ECM undergoes substantial remodeling.