Targeted RNAseq Revealed the Gene Expression Signature of Ferroptosis-Related Processes Associated with Disease Severity in Patients with Multiple Sclerosis.

Detrimental molecular processes in multiple sclerosis (MS) lead to the cellular accumulation of lipid peroxidation products and iron in the CNS, which represents the main driving force for ferroptosis. Ferroptosis is an iron-dependent form of regulated cell death, with proposed roles in neurodegeneration, oligodendrocyte loss and neuroinflammation in the pathogenesis of MS. Ferroptosis-related gene expression signature and molecular markers, which could reflect MS severity and progression, are currently understudied in humans. To tackle these challenges, we have applied a curated approach to create and experimentally analyze a comprehensive panel of ferroptosis-related genes covering a wide range of biological processes associated with ferroptosis. We performed the first ferroptosis-related targeted RNAseq on PBMCs from highly distinctive MS phenotype groups: mild relapsing-remitting (RR) (n = 24) and severe secondary progressive (SP) (n = 24), along with protein detection of GPX4 and products of lipid peroxidation (MDA and 4-HNE). Out of 138 genes, 26 were differentially expressed genes (DEGs), indicating changes in both pro- and anti-ferroptotic genes, representing a molecular signature associated with MS severity. The top three DEGs, as non-core ferroptosis genes, CDKN1A, MAP1B and EGLN2, were replicated by qPCR to validate findings in independent patient groups (16 RR and 16 SP MS). Co-expression and interactions of DEGs were presented as additional valuable assets for deeper understanding of molecular mechanisms and key targets related to MS severity. Our study integrates a wide genetic signature and biochemical markers related to ferroptosis in easily obtainable PBMCs of MS patients with clinical data and disease severity, thus providing novel molecular markers which can complement disease-related changes in the brain and undergo further research as potential therapeutic targets.

Potential beneficial effect of IFN-ß1a and ocrelizumab in people with MS during the COVID-19 pandemic.

BACKGROUND/AIM: Disease-modifying therapy (DMT) has led to added challenges in the management of people with multiple sclerosis (pwMS) during the COVID-19 era. It can reduce relapse in MS or slow down disease progression, but some DMTs can increased risk of infection. The aim of study was to evaluate risk and severity of COVID-19 in pwMS. METHODS: The examined group of pwMS were divided in group treated with IFN-ß1a, group treated with ocrelizumab and untreated group. The examination included impact of age, gender, duration of MS, type of MS, vaccination status and Expanded Disability Status Scale (EDSS) on the risk and severity of COVID-19 infection. A diagnosis of COVID-19 in pwMS was confirmed by positive polymerase-chain-reaction (PCR) or antigen test. RESULTS: Out of 207 pwMS, 82 patients were treated with ocrelizumab, 63 with IFN-ß1a, while 62 patients were untreated pwMS. The average duration of the MS was longer in the group of patients treated with ocrelizumab than in the group treated with IFN-ß1a (p < 0.05). EDSS was higher in the ocrelizumab group compared to the other two groups (p < 0.001). Untreated (more often unvaccinated) had the same COVID frequency as ocrelizumab-treated (more vaccinated, but higher EDSS). The multivariate logistic regression model indicated that administration of IFN-ß1a reduces the risk of COVID-19 infection (p = 0.001, OR = 0.381, 95% CI 0.602-0.160). The use of both DMTs, driven mainly by the IFN-ß1a effect, reduces the risk of moderate and severe COVID-19 (p < 0.05, OR = 0.105, 95% CI 0.011-0.968). CONCLUSION: This study provides evidence that IFN-ß1a can reduce the frequency of COVID-19 infection and that two DMTs, driven mainly by the IFN-ß1a effect, do not increase the risk of moderate/severe COVID-19.

Quality of sleep and fatigue in patients with the relapsing-remitting multiple sclerosis during the coronavirus disease-2019 pandemic.

ОBJECTIVE: Sleep disturbances and fatigue are frequent symptoms in multiple sclerosis patients. The aim was to assess the quality of sleep (QoS) and fatigue in patients with the relapsing-remitting multiple sclerosis (RRMS), during the coronavirus disease-2019 (COVID-19) pandemic. METHODS: The study included 67 patients with RRMS and 85 healthy control subjects. RRMS patients, who were tested in first half of 2019, were retested in April and May 2020, during the COVID-19 pandemic. We collected sociodemographic and clinical data, and also used the following questionnaires: Pittsburgh sleep quality index (PSQI), Fatigue Severity Scale (FSS), and Multiple Sclerosis Quality of Life-54 Instrument (MSQOL-54). RESULTS: The FSS score and PSQI global score were significantly higher in patients with RRMS than in the control group (p < 0.01). We noticed a statistically significant difference between the results obtained a year ago and the results during the COVID-19 pandemic in PSQI global score (p < 0.01) and all subscores. Higher disability status was an independent predictor of the worse PSQI scores. CONCLUSION: During the COVID-19 outbreak worse QoS were noticed in RRMS patients than in healthy individuals. Also, QoS of RRMS patients is more affected during the COVID-19 pandemic than in regular circumstances. High levels of sleep disturbance and fatigue in RRMS patients correlates with worse life quality, female gender, lower educational level and partner status. The results of the present study provide evidence in support of regular screening and monitoring of fatigue and QoS in this patient population, especially during the pandemic states.

Psychological status of patients with relapsing-remitting multiple sclerosis during coronavirus disease-2019 outbreak.

BACKGROUND: The coronavirus disease 2019 (COVID-19) is a global health emergency. The aim was to investigate the impact of COVID-19 pandemic on the psychological status of patients with relapsing-remitting multiple sclerosis (RRMS). METHODS: Data on the socio-demographic and clinical characteristics of 95 RRMS patients were collected. We used a self-designed questionnaire, the Multiple Sclerosis Quality of Life-54 Instrument (MSQOL-54), Hamilton scales for the assessment of anxiety (HAM-A), and depression (HAM-D). Patients who were tested one year ago were reassessed using the same questionnaires during the COVID-19 outbreak. Group of 99 healthy individuals (HC) were tested, using the same questionnaires. RESULTS: The main concerns in RRMS patients were that someone that they know could be infected with COVID-19 (78.5%), or could die due to the infection (33.8%), and the lack of specific treatment options (25.8%). The main concerns about the RRMS status were that their disease would be worse if they get infected with COVID-19 (36.4%), that they would experience some difficulties in drug availability (43.6%), that they could not go to the hospital as usual (72.4%). Results on all questionnaires were worse in RRMS patients than in HC (p<0.01). We noticed a statistically significant difference between the results obtained a year ago and the results from April 2020 in HAM-A (p<0.05). CONCLUSIONS: There is an impact of the COVID-19 pandemic on the psychological status of RRMS patients. Healthcare organizations need to provide professional therapeutic advice and psychosocial support for this population of patients during the pandemic.

The impact of betaplus program on patient treatment satisfaction with interferon beta-1b in multiple sclerosis: Multicentric cross-sectional survey in the western Balkan countries.

BACKGROUND: Long-term treatment adherence to disease-modifying drugs (DMDs) may have significant impact on clinical outcomes in multiple sclerosis (MS). It has been recently emphasized that low treatment satisfaction (TS) may be an important factor for achieving high rates of treatment adherence. Interferon (IFN) beta-1b was the first DMD approved for the treatment of MS. The aims of our study were to assess TS in subjects with relapsing-remitting (RR) MS treated with IFN beta-1b in Serbia, Montenegro and the Republika Srpska, Bosnia and Herzegovina (B&H), and additionally, to evaluate the impact of patient support program on TS and adherence. METHODS: This is a cross-sectional survey performed in order to examine TS and adherence with IFN beta-1b in seven MS centers across three countries (Serbia, Montenegro and B&H). Included in the study were 296 adult patients with RRMS treated with IFN beta-1b for at least 6 months. They were invited to complete the Treatment Satisfaction Questionnaire for Medication (TSQM). Additional two treatment adherence questions were also asked. Patient support program (Betaplus®) was available exclusively for patients in Serbia and not for those in Montenegro and the Republika Srpska, B&H. In order to assess the potential impact of this program on TSQM, we combined two groups of patients from Montenegro and B&H and compared their results with those from patients in Serbia. Statistical analysis includes multivariable linear regression analysis in order to assess the differences between three MS patients groups in terms of the TSQM scores, adjusted for potential confounders. For the evaluation of the effects of Betaplus® program, multivariable logistic regression was used, controlling for the same confounding factors. RESULTS: Each of the TSQM summary scores in all three countries implicated high level of patients' satisfaction. There was statistically significant group difference on the Effectiveness summary score (p=0.001) and the Side effects summary score (p=0.006) between the group of subjects from Serbia and the combined group of subjects from Montenegro and B&H, in favor of the former cohort. There was statistically significant group difference neither on the Convenience summary score nor on the Overall satisfaction summary score. Results of adjusted logistic regression analysis based on the availability of patient support program (dependent variable) implicate that it had the most significant impact on the Effectiveness summary score (p=0.008). According to the correlation coefficients in the total patient cohort, all TSMQ summary scores except Effectiveness significantly correlated with the decreased adherence (Side effects: p=0.037; Convenience: p=0.016; Overall satisfaction: p=0.046). CONCLUSION: TS with IFN beta-1b was high in our MS patients. Additionally, these results have demonstrated that patient support program have significant impact on TS with IFN beta-1b in the Balkan cohort of RRMS patients.

Increasing signal intensity within the dentate nucleus and globus pallidus on unenhanced T1W magnetic resonance images in patients with relapsing-remitting multiple sclerosis: correlation with cumulative dose of a macrocyclic gadolinium-based contrast agent, gadobutrol.

OBJECTIVE: To evaluate correlation between cumulative dose of gadobutrol and signal intensity (SI) within dentate nucleus and globus pallidus on unenhanced T1-weighted images in patients with relapsing-remitting multiple sclerosis (RRMS). METHODS: Dentate nucleus-to-pons and globus pallidus-to-thalamus SI ratios, and renal and liver functions, were evaluated after multiple intravenous administrations of 0.1 mmol/kg gadobutrol at 27, 96-98, and 168 weeks. We compared SI ratios based on the number of administrations, total amount of gadobutrol administered, and time between injections. RESULTS: Globus pallidus-to-thalamus (p = 0.025) and dentate nucleus-to-pons (p < 0.001) SI ratios increased after multiple gadobutrol administrations, correlated with the number of administrations (ρ = 0.263, p = 0.046, respectively) and depended on the length of administration (p = 0.017, p = 0.037, respectively). Patients receiving gadobutrol at 27 weeks showed the greatest increase in both SI ratios (p = 0.006; p = 0.014, respectively, versus 96-98 weeks). GGT increased at the end of the study (p = 0.004). CONCLUSION: In patients with RRMS, SI within the dentate nucleus and globus pallidus increased on unenhanced T1-weighted images after multiple gadobutrol injections. Administration of the same total amount of gadobutrol over a shorter period caused greater SI increase. KEY POINTS: Gadolinium deposition may occur within the human brain after multiple gadolinium contrast administrations. Increasing T1W signal intensity occurs within the dentate nucleus and globus pallidus. Increasing signal intensity may be a consequence of multiple administrations of gadobutrol. Administration of gadobutrol over a shorter period causes greater signal intensity increase.

Disease relapses in multiple sclerosis can be influenced by air pollution and climate seasonal conditions.

BACKGROUND/AIM: Environmental factors may influence the disease activity in patients with relapsing-remitting multiple sclerosis (MS). The aim of this study was to evaluate the in- fluence of air pollution and seasonal climate factors of any on number of relapses in MS patients during a consecutive 5 years of observation. METHODS: We retrospectively analyzed data of MS patients from the town of Nis, hospitalized at the Clinic of Neurology, Clinical Center Nis, Serbia, from 2005 to 2009. Climate data: mean daily sun shining; mean monthly sun shining, mean whole daily cloudiness, daily cloudiness at 7 a.m, 2 p.m. and 9 p.m. and air pollution expressed by NSR (New Source Review) were obtained from the Meteorology Observatory Nis. RESULTS: During a 5-year of observation there were 260 relapses in 101 MS patients. The number of relapses showed a significantly negative correlation with the number of days with NSR < 2 (p = -0.31; p < 0.01) and a positive correlation with the mean whole daily cloudiness (p < 0.05), mean daily cloudiness at 7 a.m. (p < 0.05) and 2 p.m. (p < 0.01). We found a significantlly positive correlation (p < 0.05) between the reduced number of relapses during the period of high vitamin D season, i.e. July-October. There was a statistically significant increase (p < 0.01) of the number of relapses during spring (x = 6.53; SD = 3.98) compared to the other three seasons. The joint presence of lower number of days with NSR < 2 during low vitamin D season (January- April) correlated with a statistically significant increase of the number of relapses in MS patients (F = 5.06, p < 0.01). CON- CLUSION: The obtained results confirmed the influence of air pollution and climate seasonal conditions on disease relapses in MS patients based on a long-term observation. Lower numbers of days with low air pollution during the periods with low vitamin D (January-April), especially with increased cloudiness at 2 p.m, induce a higher risk of MS relapses in southern continental parts of Europe.

The Prevalence of Pain in Adults with Multiple Sclerosis: A Multicenter Cross-Sectional Survey.

OBJECTIVE: Examination of prevalence, intensity and associations of pain in persons with multiple sclerosis (MS). DESIGN: Multicenter, international cross-sectional survey. SETTING: Patients were recruited from seven MS centers: in Serbia (Clinic of Neurology, Clinical Center of Serbia, Belgrade; Clinic of Neurology, Military Medical Academy, Belgrade; Clinic of Neurology, Clinical Center Kragujevac; Clinic of Neurology, Clinical Center Nis; Department of Neurology, General Hospital-Uzice), in Republic of Srpska-Bosnia and Herzegovina (Clinic of Neurology, Clinical Center Banja Luka) and in Croatia (University Department of Neurology, Sestre Milosrdnice University Hospital Center, Zagreb). SUBJECTS: Six hundred and fifty consecutive MS patients diagnosed according to the Revised McDonald criteria (2005), from the aforementioned centers, over the period of 6 months. METHODS: A semistructured questionnaire was administered during a face-to-face interview with neurologists who also performed Expanded Disability Status Scale (EDSS), the Hamilton Rating Scale for Depression (HDRS) and Hamilton Rating Scale for Anxiety (HARS). To recognize predictive factors for the presence of pain, the linear regression analysis was used. RESULTS: Lifetime prevalence of pain was 66.5% (point prevalence = 44.3%). The prevalence of the comorbidity of pain and depression was 29.1%. Older age (P < 0.001), primary-progressive MS (P = 0.034), higher EDSS score (P = 0.008), higher scores of HDRS (P < 0.001), and HARS (P < 0.001) were significantly associated with pain. Finally, in our multivariate linear regression analysis, anxiety (P < 0.001) was the independent predictor of pain. CONCLUSIONS: We confirmed high prevalence of pain, affecting approximately more than half of patients during the course of MS. Pain in MS is associated with disability, depression and, especially with anxiety, which has significant implications for treatment.

Imaging characteristics of cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL).

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) is an autosomal dominant vascular disorder. Diagnosis and follow-up in patients with CADASIL are based mainly on magnetic resonance imaging (MRI). MRI shows white matter hyperintensities (WMHs), lacunar infarcts and cerebral microbleeds (CMBs). WMHs lesions tend to be symmetrical and bilateral, distributed in the periventricular and deep white matter. The anterior temporal lobe and external capsules are predilection sites for WMHs, with higher specificity and sensitivity of anterior temporal lobe involvement compared to an external capsule involvement. Lacunar infarcts are presented by an imaging signal that has intensity of cerebrospinal fluid in all MRI sequences. They are localized within the semioval center, thalamus, basal ganglia and pons. CMBs are depicted as focal areas of signal loss on T2 images which increases in size on the T2*-weighted gradient echo planar images ("blooming effect").

Erythrocytes' antioxidative capacity as a potential marker of oxidative stress intensity in neuroinflammation.

The study is designed to assess the oxidative stress intensity in erythrocytes obtained from patients in different clinical phenotypes of neuroinflammation, defined as clinically isolated syndrome (CIS) and relapsing-remitting multiple sclerosis (RRMS). Advanced oxidation protein products (AOPP), malondialdehyde (MDA) and superoxide dismutase (SOD) activity were measured and compared with patients' clinical severity (expanded disability status scale-EDSS), radiological findings (gadolinium enhancement lesion volume-Gd+) and disease duration (DD). AOPP, MDA values and SOD activity were significantly higher in both study patients than in the control group (p < 0.05). While AOPP and MDA approached higher values in RRMS, compared to the CIS group (p > 0.05, p < 0.05, respectively), SOD activity showed higher values in CIS than in RRMS patients (p < 0.05). Both study patients with higher EDSS, higher number of total radiological lesions and longer DD, had higher AOPP and MDA content (p < 0.05, p > 0.05). SOD activity was lower in both study patients with higher EDSS, higher number of total radiological lesions and longer DD (p < 0.05, p > 0.05). There were positive correlations between AOPP and DD and EDSS in CIS patients (p < 0.01), and MDA levels and DD, EDSS and Gd+ in CIS, as well as with EDSS in RRMS patients (p < 0.01). There were negative correlations between SOD activity and DD and EDSS in both study patients (p < 0.01), as well as, between SOD activity and Gd+ in CIS patients (p < 0.01). The measured erythrocytes' biomarkers might represent one of the important biomarkers for the evaluation of the oxidative status of neuroinflammation and disease severity, especially in its early phase, defined as CIS.

Glutathione homeostasis disruption of erythrocytes, but not glutathione peroxidase activity change, is closely accompanied with neurological and radiological scoring of acute CNS inflammation.

OBJECTIVES AND METHODS: The levels of glutathione (GSH) and glutathione peroxidase (GPx) activity were measured in the erythrocytes of 50 patients with clinically isolated syndrome of CNS (CIS) and 57 patients with relapsing remitting multiple sclerosis (RRMS). RESULTS: A decrease in GSH content and GPx activity showed significance in both study groups compared to the control values (p = 0.0025 and 0.007 for GSH and p = 0.005 and 0.003 for GPx, in CIS and RRMS patients, respectively). The depletions were more pronounced in RRMS than in CIS patients (p = 0.009 for GSH and p = 0.031 for GPx). The results significantly verify the negative correlations between GSH values and clinical severity (r = -0.513, p = 0.004), radiological findings (r = -0.351, p = 0.008) and disease duration (r = -0.412, p = 0.0025) in CIS patients. The same correlations were observed in RRMS patients between GSH values and clinical severity (r = -0.498, p = 0.004) and patients' radiological features (r = -0.454, p = 0.005). No correlations were observed between GSH values and other patient characteristics, or between GPx activity and all tested patient characteristics (p > 0.01). CONCLUSIONS: The results indicate that GSH content and GPx activity both decreased below the normal range and were accompanied with neuroinflammation, but although both might have great importance in neuroinflammation development, the data presented here confirm that only GSH might serve as a marker which is closely correlated with neurological and radiological scoring of acute CNS inflammation.

The glutathione S-transferase T1 deletion is associated with susceptibility to multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) occurs as a result of interaction between genetic and environmental factors. Recent data support the view that oxidative damage is one of an early event in MS tissue injury. The safe elimination of reactive oxygen species and toxins via glutathione S-transferase (GST) pathways is required in order to protect cells against reactive oxygen-induced damage. The aim of our study was to analyze the possible association of GSTM1 and GSTT1 gene polymorphisms with the susceptibility and clinical parameters of MS, in 455 consecutive patients and 366 controls. METHODS: A multiplex polymerase chain reaction (PCR) was used to detect the deletions in GSTM1 and GSTT1 genes. RESULTS: Patients with MS had significantly higher frequency of GSTT1 null genotype compared to controls (37.36% vs. 21.86%, respectively, p<0.0001, adjusted OR 2.13 (1.56-2.90)), as well as double deletions (15.38% vs. 10.38%, respectively, p<0.05). The carriers of GSTM1 deletion had significantly earlier onset of MS compared to the wild-type carriers (28.31 ± 8.45 vs. 30.64 ± 9.30 years, respectively, p = 0.03). CONCLUSION: This study suggests the potential pathogenic role of GSTT1 deletion on MS susceptibility. There are no similar data published so far, yet this study should be replicated in other populations.

Cerebrospinal fluid and plasma oxidative stress biomarkers in different clinical phenotypes of neuroinflammatory acute attacks. Conceptual accession: from fundamental to clinic.

Oxidative stress is revealed as the main contributor in the pathophysiology of neuroinflammation. Analyzing plasma and cerebrospinal fluid (CSF) of patients with different clinical phenotypes of neuroinflammation, defined as clinically isolated syndrome (CIS), and those defined as relapsing remitting multiples sclerosis (RRMS), we tested peripheral and CNS oxidative stress intensity in these neuroinflammatory acute attacks. All obtained values changes were assessed regarding clinical and radiological features of CNS inflammation. The obtained results revealed an increase in malondialdehyde levels in plasma and CSF in CIS and RRMS patients compared to control values (p < 0.05). The obtained values were most prevailed in both study group, CIS and RRMS, in patients with severe clinical presentation (p < 0.05). Measured activities of catalase and total superoxide dismutase were higher in CIS and RRMS patients in plasma compared to control values (p < 0.05), parallel with an increased catalase activity and decrease in superoxide dismutase activity in CSF regarding values obtained in control group (p < 0.05). The positive correlations regarding clinical score were obtained for all tested biomarkers (p < 0.01). Although the positive correlations were observed in MDA levels in plasma and CSF, for both study patients, and their radiological findings (p < 0.01), and a negative correlation in plasma SOD activity and CIS patients' radiological findings (p < 0.01), no other similar correlations were obtained. These findings might be useful in providing the earliest antioxidative treatment in neuroinflammation aimed to preserve total and CNS antioxidative capacity parallel with delaying irreversible, later neurological disabilities.

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 patients with clinically isolated syndrome and relapsing remitting multiple sclerosis show different levels of advanced protein oxidation products and total thiol content in plasma and CSF.

Advanced oxidation protein products (AOPP) and total thiol (SH) groups levels in plasma and CSF were studied in a cohort of 50 clinically isolated syndrome (CIS) and 57 relapsing remittent multiple sclerosis (RRMS) patients related to 20 control group (CG) patients' values. The obtained results were compared regarding patients demographic, biochemical, clinical (EDSS) and MRI features (total T2 weighted lesions number and Gd enhancement lesion volume). Plasma and CSF AOPP levels in CIS and RRMS patients were higher than those in CG, while SH groups showed lower values compared to CG (p<0.05). Both parameters were higher in CIS than in RRMS patients (p<0.05). Related to EDSS median range, all patients were divided into those with slight or mild and those with severe clinical presentation. AOPP and SH group changes were more pronounced in both, CIS and RRMS patients with higher, compared to those with lower EDSS (p<0.05). AOPP, SH group levels and EDSS positive correlations were observed in both study groups (p<0.01). Both parameters showed the same approach regarding the median range of total T2 weighted lesions and Gd enhancement lesion volume mean values (p<0.05), but no correlation was found between AOPP and SH levels and these patients radiological characteristics (p>0.01). The data support the fact that oxidative stress is always involved in CIS and RRMS pathophysiology, but not always as a disease determinant dependent on its intensity, which might be important for new therapeutic strategies based on antioxidant approach in those patients.

INF-ß1b 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-ß1b therapy. METHODS: The study population included 15 (12 women, 3 men) untreated MS patients and 12 (10 women, 2 men) interferon-ß1b 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-ß1b 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-ß1b treatment, EDSS scores in treated patients were decreased compared to untreated ones - after 18 and 30 months (p<0.05). In interferon-ß1b 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.

Correlation of nitric oxide levels in the cerebellum and spinal cord of experimental autoimmune encephalomyelitis rats with clinical symptoms.

Experimental autoimmune encephalomyelitis (EAE) is a well-established cell-mediated autoimmune inflammatory disease of the CNS, which has been used as a model of the human demyelinating disease. EAE is characterized by infiltration of the CNS by lymphocytes and mononuclear cells, microglial and astrocytic hypertrophy, and demyelination which cumulatively contribute to clinical expression of the disease. EAE was induced in female Sprague-Dawley rats, 3 months old (300 g ± 20 g), by immunization with myelin basic protein (MBP) in combination with Complete Freund's adjuvant (CFA). The animals were divided into 7 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 level of nitric oxide (NO(·)) production was determined by measuring nitrite and nitrate concentrations in 10% homogenate of cerebellum and spinal cord. Obtained results showed that the level of NO(·) was significantly increased in all examined tissues of the EAE rats compared to the control and CFA groups. Also, AG and NAC treatment decreased the level of NO(·) in all tissues compared to the EAE group. The level of NO(·) is increased significantly in the spinal cord compared to the cerebellum. The clinical course of the EAE was significantly decreased in the EAE groups treated with AG and NAC during the development of the disease compared to EAE group and its correlates with the NO(·) level in cerebellum and spinal cord. The findings of our work suggest that NO(·) and its derivatives play an important role in multiple sclerosis (MS). It may be the best target for new therapies in human demyelinating disease and recommend the new therapeutic approaches based on a decreased level of NO(·) during the course of MS.

[Syringomyelia in demyelinating disease of the central nervous system: report of two cases].

INTRODUCTION: Syringomyelia is a cavitary extension inside the spinal cord which can be either symptomatic or congenitally-idiopathic. Syringomyelia during the course of the disease in patients presenting with clinically definite multiple sclerosis was described earlier. Syringomyelia in patients presenting with a clinically isolated syndrome suggestive of multiple sclerosis is unusual. CASE OUTLINE: We present two patients presenting with demyelinating disease of the central nervous system with syringomyelia in the cervical and thoracic spinal cord. We did not find classical clinical signs of syringomyelia in our patients, but we disclosed syringomyelia incidentally during magnetic resonance exploration. Magnetic resonance exploration using the gadolinium contrast revealed the signs of active demyelinating lesions in the spinal cord in one patient but not in the other. CONCLUSION: Syringomyelia in demyelinating disease of the central nervous system opens the question whether it is a coincidental finding or a part of clinical features of the disease. Differentiation of the significance of syringomyelia finding in these patients plays a role in the choice of treatment concept in such patients.

A case of complex arteriovenous malformation with skull destruction presented in sixth decade of life.

Intracranial AVMs are typically diagnosed before the patient has reached the age of 40 years, and a few cases have been reported of AVM with skull destruction. We described a rare case of a complex cerebral AVM with skull destruction, presented de novo in 52-year-old woman with epileptic seizures. Neuroimaging investigations revealed complex AVM in right hemisphere as well as extracranially, with signs of skull destructions, likely caused by significant involvement of feeders from external carotid artery. Neurosurgery treatment was not recommended due to morphological characteristics and drainage patterns of the AVM. EEG investigation showed discrete specific activity in correspondent area and pharmacology treatment for seizures was initiated. One year after the initial presentation patient had survived rebleeding episode witch left permanent neurology deficit. This patient considered as a rare case of complex AVM with skull destruction, presented de novo in sixth decade of life.

[Effects of alfacalcidol therapy on serum cytokine levels in patients with multiple sclerosis].

Multiple sclerosis (MS) is a consequence of genetic and environmental factors. Geographic, genetic, and biological evidence suggests that an important immunopathogenic factor might be the insufficiency of vitamin D. The aim of our study was to investigate the immunomodulatory effect of alfacalcidol, a vitamin D analogue, on cytokine levels in RRMS patients in relapse. We investigated 15 patients suffering from RRMS relapse (an RRMS group) and two control groups: one control group of healthy subjects (n=10) and a NIND group, consisting of patients with non-inflammatory neurological diseases (n=10). All of the MS patients were treated with 5 microgr/day of oral alfacalcidol for a period of five days. The serum cytokine levels of TNF-alpha, IL-10, IL-4, and IL-12 were measured in all the MS patients one day prior to and one day after therapy, and in all the control subjects (ELISA, Quantikine human immunoassay, R&D Systems, UK). Our results showed significantly lower IL-4 and IL-12 levels in the RRMS patients group compared to the N group and the NIND group (p<0.001 Mann-Whitney U-test). No significant differences in TNF-alpha and IL-10 levels were found between the groups, and there was no influence of alfacalcidol on these cytokines in RRMS patients. High doses of oral alfacalcidol induced significant increases in IL-4 and IL-12 levels in RRMS patients (p<0.001, Wilcoxon rank signed test). Therefore, there were no differences in IL-4 and IL-12 levels compared to the N group and the NIND group. Alfacalcidol therapy in RRMS patients did not provoke any side effects. Vitamin D and its analogues, such as alfacalcidol, act as immunomodulatory agents, with potential therapeutic effects for patients with multiple sclerosis.