Srdjan Ljubisavljevic

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.

The role of glutamate and its receptors in multiple sclerosis

Abstract Glutamate is an excitatory neurotransmitter of the central nervous system, which has a central role in a complex communication network established between neurons, astrocytes, oligodendrocytes, and microglia. Multiple abnormal triggers such as energy deficiency, oxidative stress, mitochondrial dysfunction, and calcium overload can lead to abnormalities in glutamate signaling. Thus, the disturbance of glutamate homeostasis could affect practically all physiological functions and interactions of brain cells, leading to excitotoxicity. Excitotoxicity is the pathological process by which nerve cells are damaged or killed by excessive stimulation by glutamate. Although neuron degeneration and death are the ultimate consequences of multiple sclerosis (MS), it is now widely accepted that alterations in the function of surrounding glial cells are key features in the progression of the disease. The present knowledge raise the possibility that the modulation of glutamate release and transport, as well as receptors blockade or glutamate metabolism modulation, might be relevant targets for the development of future therapeutic interventions in MS.

Aminoguanidine and N-acetyl-cysteine supress oxidative and nitrosative stress in EAE rat brains

Abstract Experimental autoimmune encephalomyelitis (EAE) is a well-established animal model of human multiple sclerosis (MS). We have evaluated the role of oxidative and nitrosative stress, as the causal factors in the development of EAE, responsible for the damage of cardinal cellular components, such as lipids, proteins and nucleic acids, resulting in demyelination, axonal damage, and neuronal death. EAE was induced in female Sprague-Dawley rats, 3 months old (300 ± 20 g), by immunization with myelin basic protein in combination with Complete Freunds adjuvant (CFA). The animals were divided into seven groups: control, EAE, CFA, EAE + aminoguanidine (AG), AG, EAE + N-acetyl-l-cysteine (NAC) and NAC. The animals were sacrificed 15 days after EAE induction, and the levels of nitrosative and oxidative stress were determined in 10% homogenate of the whole encephalitic mass. In EAE rats, brain NO production and MDA level were significantly increased (P < 0.001) compared to the control values, whereas AG and NAC treatment decreased both parameters in EAE rats compared to EAE group (P < 0.001). Glutathione (GSH) was reduced (P < 0.001) in EAE rats in comparison with the control and CFA groups, but increased in EAE + AG and EAE + NAC group compared to the EAE group (P < 0.01). Superoxide dismutase (SOD) activity was significantly decreased (P < 0.001) in the EAE group compared to all other experimental groups. The clinical expression of EAE was significantly decreased (P < 0.05) in the EAE groups treated with AG and NAC compared to EAE rats, during disease development. The obtained results prove an important role of oxidative and nitrosative stress in the pathogenesis of EAE, whereas AG and NAC protective effects offer new possibilities for a modified combined approach in MS therapy.

Oxidative Stress and Neurobiology of Demyelination

Despite a large amount of research which aims at defining the pathophysiology of human demyelination (i.e., multiple sclerosis), etiological bases of disease have been unknown so far. The point of intersection of all assumed etiological factors, which are mainly based upon immunological cascades, is neuroinflammation. The precise definition of the place and role of all pathogenetic factors in the occurrence and development of the disease is of crucial importance for understanding the clinical nature and for finding more effective therapeutic options. There are few studies whose results give more precise data about the role and the importance of other factors in neuroinflammation, besides immunological ones, with regard to clinical and paraclinical correlates of the disease. The review integrates results found in previously performed studies which have evaluated oxidative stress participation in early and late neuroinflammation. The largest number of studies indicates that the use of antioxidants affects the change of neuroinflammation course under experimental conditions, which is reflected in the reduction of the severity and the total reversibility in clinical presentation of the disease, the faster achieving of remission, and the delayed and slow course of neuroinflammation. Therapies based on the knowledge of redox biology targeting free radical generation hold great promise in modulation of the neuroinflammation and its clinical presentations.

Modulation of nitric oxide synthase by arginase and methylated arginines during the acute phase of experimental multiple sclerosis

We explore the nitric oxide synthase modulation by methylated arginines, asymmetric (ADMA) and symmetric (SDMA) dimethyl-l-arginine and arginase, in early phase of experimental autoimmune encephalomyelitis (EAE), the most frequently used animal model for studying the multiple sclerosis (MS), during the treatment with selective inducibile nitric oxide synthase inhibitor - aminoguanidine (AG) and oxidative scavenger N-acetyl-l-cysteine (NAC), compared to the clinical signs, continual to our previous research. The given results showed that the arginase activity was significantly increased in EAE rats compared to the healthy and AG treated EAE animals (p<0.05), and it was significantly decreased compared to the NAC treated EAE animals (p<0.05) in examined tissues. The ADMA and SDMA levels were significantly decreased in EAE untreated animals compared to the AG and NAC treated EAE animals (p<0.05). As we have reported in our previous papers, nitric oxide (NO) production, was significantly increased in examined tissues of EAE rats compared to the control group (p<0.05). In AG and NAC treated EAE group NO production was decreased in all tissues compared to untreated EAE animals (p<0.05). Also, the AG and NAC treatment of EAE rats during the development of the disease, significantly decreased the clinical score of EAE treated animals compared to EAE group. Arginase and methylated arginine derivatives, involving also NO, appear to be essential modulators of the inflammatory response in acute phase of MS. The continued research of these findings may provide a new area in the treatment of multiple sclerosis acute phase.

The reduced glutathione and S-nitrosothiols levels in acute phase of experimental demyelination--pathophysiological approach and possible clinical relevancy.

UNLABELLED Multiple sclerosis (MS) is characterized by inflammatory process associated with nitric oxide (NO) and the related species production in CNS, which can nitrosylate protein thiols and modulate their structure and functions, also reducing the CNS content of redox active compounds, such as glutathione (GSH). We have evaluated the relationships between S-nitrosothiols (RSNO) and GSH in the experimental model of MS - experimental autoimmune encephalomyelitis (EAE), during the treatment with inducible NO synthase inhibitor - aminoguanidine (AG) and thiol donor molecule - N-acetyl-L-cysteine (NAC). MATERIAL AND METHODS EAE was induced by myelin basic protein, dissolved in phosphate-buffered saline (PBS), emulsified in the complete Freunds adjuvant (CFA) followed by injections of Pertussis toxin. Animals assigned to the control (PBS), EAE, CFA, EAE+AG, AG, EAE+NAC and NAC groups were scored daily for the clinical signs of EAE. RSNO and GSH were evaluated in whole encephalitic mass and cerebellum. RESULTS RSNO concentration was increased in EAE-untreated animals compared to the AG and NAC-treated EAE animals (p<0.05). Also, during the treatment with AG and NAC, GSH concentration was increased compared to the untreated animals (p<0.05). The EAE clinical signs were reduced in EAE-treated animals compared to the other groups (p<0.05). CONCLUSION The findings of our work suggest a potential role of RSNO and GSH in early clinical presentation of experimental MS, that might be also useful as predictive parameters for MS treatment directed to increased GSH and thiol pool in CNS.

INF-β1b therapy modulates L-arginine and nitric oxide metabolism in patients with relapse remittent multiple sclerosis.

OBJECTIVE The scope of this study is the examination of NO(2)+NO(3), 3-nitrotyrosine (3-NT), S-nitrosothiols (RSNO), arginase activity and asymmetric (ADMA) and symmetric (SDMA) dimethyl-L-arginine concentrations in plasma of MS patients during interferon-β1b therapy. METHODS The study population included 15 (12 women, 3 men) untreated MS patients and 12 (10 women, 2 men) interferon-β1b treated MS patients with clinically definite relapsing MS (McDonalds criteria) for at least 1 year and a baseline EDSS score of 1.0 to 3.5 inclusive. Patients were treated with 250 μg IU interferon-β1b s.c. every second day during 30 months. The disease course was evaluated using correlations between baseline EDSS score and relapse rates in both groups. RESULTS During interferon-β1b treatment, EDSS scores in treated patients were decreased compared to untreated ones - after 18 and 30 months (p<0.05). In interferon-β1b treated MS patients, NO(2)+NO(3), 3-NT and RSNO plasma concentrations were significantly lower (p<0.05), while arginase activity, ADMA and SDMA levels were significantly increased (p<0.05) during the therapy, compared to the baseline levels in treated patients. CONCLUSION The investigated parameters may be the new biomarkers, providing information for the therapeutic approach and valuable in clinical monitoring.

Neuroinflammation and demyelination from the point of nitrosative stress as a new target for neuroprotection.

Abstract The role of nitrosative stress in the early pathogenesis of neuroinflammation and demyelination is undoubtedly wide. This review summarizes and integrates the results, found in previously performed studies, which have evaluated nitrosative stress participation in neuroinflammation. The largest number of studies indicates that the supply of nitrosative stress inhibitors has led to the opposite clinical effects in experimental studies. Some results claim that attributing the protective role to nitric oxide, outside the total changes of redox oxidative processes and without following the clinical and paraclinical correlates of neuroinflammation, is an overrated role of this mediator. The fact is that the use of nitrosative stress inhibitors would be justified in the earlier phases of neuroinflammation. The ideal choice would be a specific inducible nitric oxide synthase (iNOS) inhibitor, because its use would preserve the physiological features of nitric oxide produced by the effects of constitutive NOS. This review discusses the antinitrosative therapy as a potential mode of therapy that aims to control neuroinflammation in early phases, delaying its later phases, which are accompanied with irreversible neurological disabilities. Some parameters of nitrosative stress might serve as surrogate biomarkers for neuroinflammation intensity and its radiological and clinical correlates.

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.

The Role of Matrix Metalloproteinase 3 and 9 in the Pathogenesis of Acute Neuroinflammation. Implications for Disease Modifying Therapy.

Matrix metalloproteinases (MMPs) are proteolytic enzymes that are involved in a variety of physiological and pathological processes, including those in CNS. In this study, plasma values of MMP-3 and MMP-9 have been compared in clinically isolated syndrome (CIS) and relapsing-remitting multiple sclerosis (RRMS) patients during their acute attacks, in relation to the biological activity of disease. Therefore, we compared the MMPs plasma values regarding Expanded Disability Status Scale (EDSS), progression index of disease (PID), acute brain lesion volume seen on magnetic resonance imaging (MRI) and index of blood-brain barrier (BBB) permeability destruction. The obtained results demonstrated higher plasma values of MMPs in both study groups than control values (p < 0.05). No statistical significances have been detected comparing the obtained values of both enzymes between CIS and RRMS group (p > 0.05). In both CIS and RRMS groups, the patients with higher EDSS showed higher MMPs plasma values (p < 0.05). The MMPs values were also significantly higher in both study patients with higher total number comparing to those with lower number of MRI brain lesion (p < 0.05) (beyond MMP-3 in RRMS). All obtained correlations, between MMPs and EDSS, PID, volume of MRI Gd-enhancement brain lesions, and index of BBB permeability, were positive (p < 0.05.) This study demonstrates alterations of both tested MMPs with closed correlation with the disease biological activity. Although MMPs are being implicated in the pathogenesis of acute neuroinflammation, the MMPs modulation might be useful in the future design of disease modifying therapy with the specific target profile.