Tumor necrosis factor-alfa and interleukin-4 in cerbrospinal fluid and plasma in different clinical forms of multiple sclerosis

Background/Aim. Multiple sclerosis (MS) is an immunemediated central nervous system disease characterized by inflammation, demyelination and axonal degeneration. Cytokines are proven mediators of immunological process in MS. The aim of this study was to investigate whether there is a difference in the production of the tumor necrosis factor alpha (TNF-alpha) and interleukin-4 (IL-4) in cerebrospinal fluid (CSF) and plasma in the MS patients and the controls (other neurological non-inflammatory diseases) and to determine a possible difference in these cytokines in plasma and CSF in different clinical forms of MS. Methods. This study involved 60 consecutive MS patients - 48 patients with relapsing-remitting MS (RRMS) and 12 patients with secondary progressive MS (SPMS). The control group consisted of 20, age and sex matched, nonimmunological, neurological patients. According to the clinical presentation of MS at the time of this investigation, 34 (56.7%) patients had relapse (RRMS), 14 (23.3%) were in remission (RRMS), while the rest of the patients, 12 (20.0%), were SPMS. TNF-alpha and IL-4 concentrations were measured in the same time in CSF and plasma in the MS patients and the controls. Extended disability status score (EDSS), albumin ratio and IgG index were determined in all MS patients. Results. The MS patients had significantly higher CSF and plasma levels of TNF-alpha than the controls (p < 0.001 for both samples). IL-4 CSF levels were significantly lower in the MS patients than in the controls (p < 0.001), however plasma levels were similar. The patients in relapse (RRMS) and with progressive disease (SPMS) had higher concentrations of CSF TNF-alpha levels than the patients in remission (p < 0.001). IL-4 CSF levels in relapse (RRMS) and SPMS groups were lower than in the patients in remission. The patients in remission had an unmeasurable plasma TNF-alpha level and the patients with SPMS had significantly lower IL-4 levels in plasma than the patients in relapse and remission (p < 0.001). The only significant correlation between cytokine level with either EDSS, or albumin ratio, or IgG index, was found between CSF TNF-alpha levels and albumin ratio in the patients with relapse (R square = 0.431, p < 0.001). Conclusion. According to the obtained data MS relapse was characterized by high concentrations of TNF-alpha in CSF and plasma and low concentrations of IL-4 in CSF. Remission was characterized by high concentrations of IL-4 and low concentrations of TNF-alpha both in CSF and plasma. SPMS was characterized with lower concentrations of TNF-alpha and IL-4 compared to relapse, both in CSF and plasma

Early-onset ischaemic stroke in a patient with the novel F2 c.1824C>T gene variant and PAI-1 4G/4G, MTHFR 677TT genotype

Introduction. Ischemic stroke (IS) is a heterogeneous dis-order caused by several genetic and environmental risk factors. It was suggested that coagulation disorders cause 1-4% of cases with IS, especially in patients with early onset of IS. Case report. We describe a case of a young adult male who developed an unprovoked IS. Biochemical, immunological, and thrombophilia screening, as well as DNA sequencing, were performed in order to reveal molecular pathology underlying the stroke of the patient. Thrombophilia testing showed that patient was a homozygous carrier for PAI-1 4G/5G and MTHFR C677T mutations. Additional genetic analysis revealed the presence of the recently reported F2 c.1824C>T gene variant, located in the last exon of the pro-thrombin gene and has previously been shown to cause hy-perprothrombinemia, hypofibrinolysis, and altered fibrin clot phenotype. Conclusion. Our results suggest that the newly reported F2 c.1824C>T gene variant might have a synergistic effect with PAI 4G/4G and MTHFR 677TT genotype in the formation of altered fibrin clot phenotype characterized by thin, densely packed fibrin fibers, which makes clot less susceptible to fibrinolysis and greatly in-creases the risk for early ischemic stroke onset.Ishemijski moždani udar (IMU) je heterogeni poremećaj koji može biti uzrokovan genetskim faktorima rizika i faktorima sredine. Poremećaji koagulacije mogu biti uzročnici u 1-4% slučajeva IMU, naročito kod bolesnika kod kojih se IMU dogodi u mlađem životnom dobu. Prikaz bolesnika. Prikazan je slučaj bolesnika koji je u mlađem životnom dobu razvio IMU nepoznatog uzroka. Urađeni su biohemijski, imunološki i testovi za trombofiliju kao i sekvenciranje DNK sa ciljem da se utvrdi molekularna patologija koja je mogla biti u osnovi moždanog udara kod tog bolesnika. Testovima za trombofiliju utvrđeno je da je bolesnik homozigotni nosilac mutacija PAI-1 4G/5G i MTHFR C677T. Dodatnom genetičkom analizom otkriveno je prisustvo nedavno opisane F2 c.1824C>T genske varijante, koja se nalazi u poslednjem egzonu gena za protrombin i za koju je prethodno pokazano da izaziva hiperprotrombinemiju, hipofibrinolizu i izmenjeni fenotip fibrinskog ugruška. Zaključak. Naši rezultati ukazuju na to da bi nova F2 c.1824C>T genska varijanta mogla imati sinergistički efekat sa PAI 4G/4G i MTHFR 677TT genotipom u nastanku fibrinskog ugruška sa izmenjenim fenotipom, koji se odlikuje tankim, gusto upakovanim fibrinskim vlaknima, što čini ugrušak manje podložnim fibrinolizi i povećava rizik od nastanka IMU u ranijem životnom dobu

Rizik razvoja multiple skleroze povezan sa oksidativnim stresom

Background: Multiple sclerosis (MS) is characterized by inflammation, demyelination and axonal degeneration. Oxidative stress (OS) plays a significant role in the pathogenesis of the disease. The aim of the study was to examine the association between OS and smoking on the MS development. Methods: The study included 175 patients with relapsing- remitting multiple sclerosis (RRMS) (76 males, 99 females) and 254 healthy subjects (81 males and 173 females). Oxidative stress biomarkers in serum, Total Antioxidant Status (TAS) and Total Oxidative Status (TOS) were determined spectrophotometrically. Oxidative Stress Index (OSI) was calculated as the ratio of TOS and TAS. Urinary 8-oxo- 7,8-dihydro-2’-deoxyguanosine were determined by HPLC-MS/MS and expressed as 8-oxodG/creatinine. Results: In females with RRMS were higher TOS, OSI and 8-oxodG/creatinine than in females in control group. The group of males with RRMS had lower level of TAS than the males in control group. Higher levels of 8-oxodG/creatinine was obtained in active, passive and former smokers with RRMS than in control group with the same exposition to tobacco smoke. Independent predictors of MS are passive smoking, increased OSI and increased levels of urinary 8-oxodG/creatinine. Conclusions: Our results demonstrate that the OS parameters should be included in the assessment of the risk for MS development. Due to the more sensitivity to oxidative stress, females may be at higher risk of MS development. This data indicates the importance of introducing the antioxidant therapy as a complementary treatment in patients with RRMS.Uvod: Multipla skleroza (MS) se karakteriše upalom, demijelinizacijom i degeneracijom aksona. Oksidativni stres (OS) igra značajnu ulogu u patogenezi bolesti. Cilj studije je bio da se ispita povezanost OS i pušenja na razvoj MS. Metode: Studija je obuhvatila 175 pacijenata sa relapsnoremitentnom multiplom sklerozom (RRMS) (76 muškaraca, 99 žena) i 254 zdrava ispitanika (81 muškarac i 173 žene). Biomarkeri oksidativnog stresa u serumu, ukupni antioksidativni status (TAS) i ukupni oksidativni status (TOS) su određeni spektrofotometrijski. Indeks oksidativnog stresa (OSI) je izračunat kao odnos TOS i TAS. Urinarni 8-okso7,8-dihidro-2'-deoksiguanozin je određen HPLC-MS/MS i izražen kao 8-oksoG/kreatinin. Rezultati: Kod žena sa RRMS bili su viši TOS, OSI i 8okodG/kreatinin nego kod žena u kontrolnoj grupi. Grupa muškaraca sa RRMS imala je niži nivo TAS od muškaraca u kontrolnoj grupi. Veći nivoi 8-okodG/kreatinina su dobijeni kod aktivnih, pasivnih i bivših pušača sa RRMS nego u kontrolnoj grupi sa istom izloženošću duvanskom dimu. Nezavisni prediktori MS su pasivno pušenje, povećan OSI i povećani nivoi 8-okodG/kreatinina u urinu. Zaključak: Naši rezultati pokazuju da parametre OS treba uključiti u procenu rizika za razvoj MS. Zbog veće osetljivosti na oksidativni stres, žene mogu biti izložene većem riziku od razvoja MS. Ovi podaci ukazuju na značaj uvođenja antioksidativne terapije kao komplementarnog lečenja kod pacijenata sa RRMS

Gene expression of chemokines CX3CL1 and CXCL16 and their receptors, CX3CR1 and CXCR6, in peripheral blood mononuclear cells of patients with relapsing-remitting multiple sclerosis - a pilot study

Background/Aim. In vitro and in vivo studies show that CX3CL1 and CXCL16 chemokines and their specific receptors, CX3CR1 and CXCR6, respectively, mediate mechanism of neuroinflammation during the pathogenesis of multiple sclerosis (MS). The aim of this study was to investigate relative messenger ribonucleic acid (mRNA) levels of CX3CL1, CXCL16, CX3CR1 and CXCR6 in peripheral blood mononuclear cells, as potential molecular markers of relapsing-remitting (RR) MS. Methods. The study included 43 unrelated RR MS patients, 20 of them with clinically active disease (relapse) and 23 with clinically stable disease (remission), and 28 unrelated healthy subjects as controls. Real-time polymerase chain reactions (PCR) were performed using TaqMan? gene expression assays. Relative expression (mRNA) level of each target gene in each sample of peripheral blood mononuclear cells was calculated as the mean normalized expression. Results. The levels of CX3CR1 mRNA were significantly higher in clinically active RR MS patients compared to controls [fold change = 1.38, p (Mann-Whitney U test) = 0.009], and significantly lower in clinically stable vs active RR MS patients [fold change = - 1.43, p (t-test) = 0.03]. Stable RR MS patients had significantly higher CXCL16 mRNA levels than controls [fold change = 1.33, p (Mann-Whitney U test) = 0.006]. A trend of increased CXCR6 gene expression was found in active RR MS patients compared to controls [fold change = 1.23, p (Mann-Whitney U test) = 0.08]. In either active or stable RR MS patients there were no significant correlations of the clinical parameters with expression levels of the target genes. Conclusion. The current results show that increased CX3CR1 mRNA levels in peripheral blood mononuclear cells could represent a proinflammatory molecular marker of clinically active RR MS

Small internal jugular veins with restricted outflow are associated with severe multiple sclerosis: a sonographer-blinded, case-control ultrasound study

Background: Recent evidence has indicated an association between chronic cerebrospinal venous insufficiency (CCSVI) and multiple sclerosis. Small internal jugular veins (IJVs) (with a cross-sectional area of less than 0.4 cm(2)) have been previously described as difficult to catheterize, and their presence may potentially affect cerebrospinal venous drainage. In this blinded extracranial color-Doppler study we had two principal aims: first, to assess prevalence of CCSVI among Serbian MS patients compared to healthy controls; and second, to assess prevalence of small IJVs (with a CSA LT = 0.4 cm(2)) among MS patients and controls. Methods: The sixty seven unrelated patients with clinical isolated syndrome (CIS), relapsing-remitting (RR), secondary progressive (SP) and primary progressive (PP) multiple sclerosis and 21 healthy controls were examined by high-resolution color-Doppler. Results: The ultrasonographic criteria of CCSVI (according to Zamboni) were positive in 11.9% of the patients and in none of the control subjects. The CCSVI-positive patients had significantly longer disease durations and were significantly more disabled (measured by their Expanded Disability Status Scale (EDSS) and Multiple Sclerosis Severity Score (MSSS) scores), but after adjustment for gender and disease duration, CCSVI was not an independent risk factor for multiple sclerosis severity. The small IJVs were found in 28.4% of the patients and 28.6% of the controls. The patients with small IJVs were associated with decreased venous outflow from the brain and presented with longer disease durations and significantly higher EDSS and MSSS scores compared to patients without small IJVs. A multivariate logistic regression analysis adjusted for gender and disease duration showed that small IJV is an independent factor associated with multiple sclerosis severity (EDSS GT = 6) (adjusted OR = 8.9, 95% CI: 1.8-45.6, p = 0.007). Among patients with small IJVs the 36.84% were also CCSVI positive. Conclusions: Both, CCSVI and small IJVs seem to influence or follow MS severity, but only small IJVs turned out to be an independent factor in this study. Thus, small IJVs with restricted outflow, which might be aspects of CCSVI different from the criteria originally described by Zamboni, emerge as a cofactor in the multifactorial pathophysiology of multiple sclerosis

FADS2 gene variant rs174593 is associated with multiple sclerosis

Introduction: The hallmark pathogenic mechanisms of multiple sclerosis (MS) are proposed to be associated with long chain polyunsaturated fatty acids(LC-PUFA)-mediated neuroinflammation, through LC-PUFA-derived pro- and anti-inflammatory eicosanoids. Variants in genes coding for fatty acid desaturases (FADS), the key enzymes in LC-PUFA biosynthesis from essential fatty acids, are associated with changesin circulating LC-PUFA levels. The aim of thisstudy wasto investigate the FADS2 intronic variants, rs174576 (C/A), rs174593 (T/C) and rs174616 (G/A), in association with MS. Methods: The study involved 124 patients with relapsing-remitting form of MS and 83 healthy control subjects. The FADS2 gene variants were detected using TaqMan® SNP genotyping assays. Analysis of allele and genotype distributions in patients and controls was done by using the chi-square test. Results: According to the model of dominant effect of allele, genotypes containing the alternative, C, allele of FADS2 rs174593 variant were significantly less frequent in MS patients than in controls (MS: TT=57,26%, TC+CC=42,74%; controls: TT=42,17%, TC+CC=57,83%; p=0,03). In addition, the frequency of rs174593 C allele was significantly lower in patients, compared to controls (MS: T=0,76, C=0,24; controls: T=0,67, C=0,33; p=0,04). The frequency distributions of rs174576 and rs174616 alleles and genotypes were not significantly different between the study groups (p>0,05). Conclusion: The obtained resultssupply a rationale for further investigation of the association of FADS2 rs174593 with circulating LC-PUFA levels, in the context of MS. The genotype-LC-PUFA phenotype association could provide guidelinesfor personalized LC-PUFA supplementation, to potentially ameliorate the disease course and improve the effectiveness of therapyThe Second Congress of Molecular Biologists of Serbia; October 6-8, Belgrade, 2023

Insulin resistance in drug naive patients with multiple sclerosis

Background/Aim. Due to the fact that there is a relatively small number of data related to systemic insulin abnormalities in the multiple sclerosis (MS), the main objective of our study was to determine whether a dysbalance of glucose and insulin metabolism exist in patients with natural course of MS. Our hypothesis was that the metabolic disorder that characterizes state of the insulin resistance (IR) and reduced insulin sensitivity (IS) in untreated patients with MS could play a role in disease progression and degree of functional disability. Methods. The study included 31 patients with relapsing-remitting (RR) MS and 14 healthy controls from the same geographic area matched by age, ethnicity and number of smokers. The glucose tolerance, IS, and IR were examined using an oral glucose tolerance test (OGTT) and using basal plasma glucose and insulin levels. The functional disability and disease progression were evaluated by the Expanded Disability Status Scale (EDSS) and Multiple Sclerosis Severity Score (MSSS). Results. The MS patients tolerated glucose equally well as the healthy controls. Basal concentrations of insulin were significantly higher in the MS group p LT 0.05), as well as insulin plasma level 30 min after oral glucose load (p LT 0.01). The patients with MS had significantly higher values of homeostasis model assessment indexes of IR (HOMA-IR) (p = 0.027; p = 0.028). The percentage of IS (HOMA2 % S) and whole body IS index (ISI Matsuda) showed significantly lower values in the MS patients than in the controls (p = 0.005; p = 0.001). The insulinogenic index in the first 30 min of OGTT was significantly higher in MS patients (p = 0.005). The measures of functional disability and MS progression did not correlate significantly with the investigated parameters of IR and IS indexes. Conclusion. This study demonstrates for the first time the existence of hyperinsulinemia, reduced insulin sensitivity and normal glucose tolerance that indicate the initial phase of IR in the natural course of MS. Additional research is necessary in order to define the mechanisms of occurrence and the impact of IR on the complex pathophysiological processes in MS

Angiotensin II receptor type 2 (AT2R) -1332 A/G gene polymorphism as a risk factor for multiple sclerosis

Multiple sclerosis (MS) is a complex inflammatory,demyelinating disease of central nervous system (CNS). All the essen-tial components of the renin-angiotensin system (RAS) are presentedin the mammalian brain. The angiotensin II (Ang II), biologicallyactive octapeptide is not only a vasoconstrictor, but also a pro-inflam-matory factor. Many of the classical and of the hypothetical functionsof brain Ang II are mediated by stimulation of AT1 receptors (AT1R).Brain AT2 receptors (AT2R) are highly expressed during development.In the adults, AT2R are restricted to areas predominantly involved inthe process of sensory information. The AT2R1332 A/G polymorph-ism was proposed to influence AT2R protein expression, and is themost studied polymorphism in this gene, in other diseases. Recently,the striking appearance of the RAS in MS brain was described.However, the role of AT2R remains to be clarified. Thus, the aim ofour study was to establish if there is an association between AT2R1332 A/G gene polymorphism and predisposition of MS Methods:Subjected group consisted of 122 female and 70 malepatients with MS and 75 female and 50 male controls from populationof Serbia. Genotyping was done by PCR and restriction digestion withEcoRI enzyme.Results:The genotype and allele frequencies for AT2R1332A/Ggene polymorphism are analyzed separately in females and males,since this gene is located on X chromosome. We detected significantoverrepresentation of1332A/G AA genotype (OR 1.6, 95% CI:1.0–2.7, p<0.05) in female patients with MS compared to female controls.In hemizygous males we didn’t found any difference between patientsand controls.Conclusion:The role of RAS genes in MS was neglected untilrecently. Than, it was shown that the role of RAS in the CNS isbeyond the regulation of cardiovascular function. Until now AT2R(1332A/G) gene polymorphism was widely studied and associatedwith hypertension and other vascular disease. Until now, there wereno studies concerning role of Ang II receptor polymorphisms in MS.This study suggest possible role of AT2R in MS. Further studies areneeded to elucidate this result.ECTRIMS : The 26th Congress of the European Committee for Treatment and Research in Multiple Sclerosis : Book of abstract

Association of circadian rhythm genes ARNTL/BMAL1 and CLOCK with multiple sclerosis

Prevalence of multiple sclerosis varies with geographic latitude. We hypothesized that this fact might be partially associated with the influence of latitude on circadian rhythm and consequently that genetic variability of key circadian rhythm regulators, ARNTL and CLOCK genes, might contribute to the risk for multiple sclerosis. Our aim was to analyse selected polymorphisms of ARNTL and CLOCK, and their association with multiple sclerosis. A total of 900 Caucasian patients and 1024 healthy controls were compared for genetic signature at 8 SNPs, 4 for each of both genes. We found a statistically significant difference in genotype (ARNTL rs3789327, P = 7.5.10(-5); CLOCK rs6811520 P = 0.02) distributions in patients and controls. The ARNTL rs3789327 CC genotype was associated with higher risk for multiple sclerosis at an OR of 1.67 (95% CI 1.35-2.07, P = 0.0001) and the CLOCK rs6811520 genotype CC at an OR of 1.40 (95% CI 1.13-1.73, P = 0.002). The results of this study suggest that genetic variability in the ARNTL and CLOCK genes might be associated with risk for multiple sclerosis