Paraoxonase 1 activity in multiple sclerosis patients during mitoxantrone therapy.

OBJECTIVES: It has been implicated in many studies that reactive oxygen species play a role in the development of demyelination in multiple sclerosis (MS). Paraoxonase 1 (PON1) is an antioxidant enzyme that protects cell membranes against oxidative modification. Mitoxantrone is a cytotoxic drug approved for the treatment of MS with adverse effects associated potentially with an increased level of oxidative stress. The aim of this study was to assess the influence of mitoxantrone therapy on PON1 activity in patients with MS. METHODS: A studied group included 26 patients with secondary progressive MS, 16 women and 10 men. The blood was collected before the beginning of the therapy as well as after 6 and 12 months. Patients were receiving mitoxantrone every 12 weeks. Serum PON1 activity was assayed using two synthetic substrates: paraoxon and phenyl acetate. RESULTS: Paraoxonase 1 activity toward paraoxon and phenyl acetate and lipid profile did not change significantly in patients receiving mitoxantrone. CONCLUSIONS: Mitoxantrone therapy does not influence PON1 activity.

A randomized, double-blind, placebo-controlled, parallel-group, enriched-design study of nabiximols* (Sativex(®) ), as add-on therapy, in subjects with refractory spasticity caused by multiple sclerosis.

BACKGROUND: Spasticity is a disabling complication of multiple sclerosis, affecting many patients with the condition. We report the first Phase 3 placebo-controlled study of an oral antispasticity agent to use an enriched study design. METHODS: A 19-week follow-up, multicentre, double-blind, randomized, placebo-controlled, parallel-group study in subjects with multiple sclerosis spasticity not fully relieved with current antispasticity therapy. Subjects were treated with nabiximols, as add-on therapy, in a single-blind manner for 4weeks, after which those achieving an improvement in spasticity of ≥20% progressed to a 12-week randomized, placebo-controlled phase. RESULTS: Of the 572 subjects enrolled, 272 achieved a ≥20% improvement after 4weeks of single-blind treatment, and 241 were randomized. The primary end-point was the difference between treatments in the mean spasticity Numeric Rating Scale (NRS) in the randomized, controlled phase of the study. Intention-to-treat (ITT) analysis showed a highly significant difference in favour of nabiximols (P=0.0002). Secondary end-points of responder analysis, Spasm Frequency Score, Sleep Disturbance NRS Patient, Carer and Clinician Global Impression of Change were all significant in favour of nabiximols. CONCLUSIONS: The enriched study design provides a method of determining the efficacy and safety of nabiximols in a way that more closely reflects proposed clinical practice, by limiting exposure to those patients who are likely to benefit from it. Hence, the difference between active and placebo should be a reflection of efficacy and safety in the population intended for treatment.

CT volume/density ratio as the marker of ischaemic brain injury.

OBJECTIVES: We believe that the CT volume/density ratio (VDR) of infarcted area reflects the degree of brain tissue damage during ischaemic stroke (IS). PATIENTS AND METHODS: Forty six patients with IS were prospectively enrolled into the study. CT scan was performed on days 1 and 10 of hospitalization. S100BB serum level, gelatinase activities (MMP-2 and MMP-9) and neurological examination (NIHSS) were performed on days 1, 5 and 10 of IS. After 3 months, 42 patients were examined by functional disability scales: Barthel index (BI) and modified Rankin scale (mRS). RESULTS: The VDR of ischaemic focus correlated well with the average S100BB serum level, MMP-9 serum activity and NIHSS score. The weak but statistically significant relationships were noticed between the VDR vs BI and mRS estimated 3 months after stroke. CONCLUSION: VDR reflects well the damage ratio of brain tissue during IS. In addition, the study underlines the relationship between VDR vs patients' neurological status and disability after IS.

Total tau and S100b proteins in different types of multiple sclerosis and during immunosuppressive treatment with mitoxantrone.

PURPOSE: Brain-specific proteins are biochemical markers of neurodegeneration. The aim of this study was to estimate the role of biomarkers in neuronal and glial damage as a potent marker of efficiency of immunosuppressive treatment. MATERIAL AND METHODS: The levels of total Tau protein (tTau) and S100b protein were measured using the ELISA method in serum and cerebrospinal fluid (CSF) of 30 patients with RRMS, 24 patients with SPMS and 30 healthy subjects. Additionally, serum levels of tTau and S100b were assayed every 6 months during the 24-month mitoxantrone therapy. RESULTS: In CSF and serum of patients with MS, both tTau and S100b were increased compared to control group; however, no significant difference was found between respective MS types. In serum of mitoxantrone-treated patients, both proteins showed to decrease after 24 months, yet the difference was statistically significant only for S100b. CONCLUSIONS: CSF levels of tTau and S100b are elevated in patients with MS and can reflect an axonal and glial pathology. Measurement of serum concentrations of S100b may be useful for monitoring immunosuppressive therapy and may support clinical assessment. In contrast, tTau concentration did not prove to be a useful marker of mitoxantrone therapy.

Impact of cladribine on soluble adhesion molecules in multiple sclerosis.

BACKGROUND: Soluble forms of vascular cell adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1) and E-Selectin play a role in the regulation of blood-brain barrier damage and represent markers of the clinical course of multiple sclerosis (MS) and magnetic resonance imaging activity. We determined sICAM, sVCAM and sE-Selectin concentrations in the cerebrospinal fluid (CSF) and serum of patients with remitting-relapsing multiple sclerosis before and after cladribine treatment as well as in a control group. METHODS: We examined 17 patients diagnosed according to McDonald's criteria. Thirteen healthy age-matched subjects served as controls. The ELISA method was used to measure sICAM-1, sVCAM-1 and sE-Selectin. RESULTS: The concentration of sICAM and sE-Selectin decreased in sera (difference between patients and controls was statistically significant, in the former P < 0.04, in the latter P < 0.0003) but not in the CSF of MS patients after cladribine treatment. CONCLUSIONS: The reduction in sICAM and sE-Selectin concentrations after cladribine treatment indicates an immuno-suppressive effect of the drug. The changes in levels of sICAM and sE-Selectin after cladribine treatment reflect disease activity and indicate a reduction in the inflammatory reaction.

Effect of parenteral cladribine on relapse rates in patients with relapsing forms of multiple sclerosis: results of a 2-year, double-blind, placebo-controlled, crossover study.

OBJECTIVE: This randomized, 2-year, double-blind, placebo-controlled, crossover study evaluated cladribine for relapsing forms of multiple sclerosis. PATIENTS: (n = 84) received seven 5-day courses of subcutaneous cladribine at 5 mg/day (group A) or placebo (group B) in year 1; treatment was reversed in year 2. RESULTS: Cladribine was well tolerated and associated with a favorable safety profile. Mean Expanded Disability Status Scale scores remained stable. In group A, mean relapse rates were 0.15 in year 1 (cladribine) and 0.42 in year 2. In group B, relapse rates were 0.61 in year 1 and 0.50 in year 2 (cladribine). PATIENTS required fewer steroid courses during cladribine periods. The therapeutic efficacy of cladribine was associated with a sustained reduction in lymphocyte count.

Paraoxonase 1 activity in different types of multiple sclerosis.

BACKGROUND: Paraoxonase 1 (PON1) is an antioxidant enzyme bound to plasma high-density lipoproteins and is also present in the brain. OBJECTIVE: The aim of this study was to estimate the activity of PON1 in patients with different types of MS. METHODS: The PON1 activity toward paraoxon and phenyl acetate and lipid profile was examined in 40 relapsing-remitting (RR) patients in relapse, in 42 RR patients in remission, in 55 progressive MS patients and in 40 healthy individuals. RESULTS: PON1 activity did not differ in MS patients compared to control group. PON1 activity in relapse was significantly lower in comparison to the other MS groups. Hypercholesterolemia was observed in MS patients. CONCLUSION: PON1 activity does not change in the course of stable and progressive type of MS and is decreased by the relapse of MS.

Expression of glutamate transporters GLT-1 and GLAST in different regions of rat brain during the course of experimental autoimmune encephalomyelitis.

Demyelination and oligodendroglial cell death accompanied by axonal injury are dominating features of multiple sclerosis (MS) a chronic demyelinating disease of the CNS. Accumulation of extracellular glutamate, observed during MS, is implicated in excitotoxic injury of nerve and oligodendroglial cells as a result of over-activation of glutamate receptors. The appropriate concentration of extracellular glutamate is maintained by glutamate transporters, the most predominant of which is glial transporter GLT-1 (excitatory amino acid transporter (EAAT) 2). The aim of this study is to determine the time-course of GLT-1 and glutamate-aspartate transporter (GLAST) expression in forebrain and cerebellum of rats subjected to experimental autoimmune encephalomyelitis (EAE). Our findings revealed that: (1) GLT-1 mRNA and to a lower extent GLAST mRNA are overexpressed in forebrain and cerebellum of EAE rats (2) expression of GLT-1 transporter mRNA shows a similar temporal pattern throughout the course of EAE in both structures examined, and is closely correlated with the appearance of neurological symptoms; and (3) the expression of GLT-1 and GLAST protein does not mirror mRNA changes during EAE and exhibits a differential spatial pattern. The protein levels of GLT-1 in cerebellum and GLAST in both structures are significantly reduced just before the acute phase and later during the recovery. The results imply that transcriptional up-regulation of the GLT-1 gene occurs early in both the forebrain and the cerebellum of the EAE rat model. This up-regulation is associated with the severity of symptoms but tends to precede the onset of maximal neurological deficits. The observations confirm the involvement of glutamate in the pathogenesis of EAE and provide an indication of the protective role of this glutamate transporter. However, changes in protein expression of both transporters suggest the existence of post-translational disturbances or the influence of regulating factors connecting with EAE conditions that may lead to the insufficient protection against glutamate excitotoxicity.

Cerebrospinal fluid brain specific proteins in relation to nitric oxide metabolites during relapse of multiple sclerosis.

This study investigated the cerebrospinal fluid (CSF) levels of ferritin, S100B as biomarkers for glial activation and NfH(SM135)--a biomarker of axonal damage--in relation to nitric oxide (NO) metabolites: nitrate and nitrite (NOx) during acute multiple sclerosis (MS) relapse. Thirty-four relapsing-remitting MS (RR-MS) patients during acute relapse and 12 controls were enrolled. Patients were assessed on Expanded Disability Status Scale (EDSS) and underwent lumbar puncture within two weeks following relapse. Twenty patients were available for further follow-up and were assessed on EDSS 6-8 weeks since the relapse onset. The CSF NOx (P<0.0001), NfH(SM135) (P=0.01) and S100B (P=0.009) but not ferritin (P>0.05) were significantly raised in MS group. There was a significant correlation between CSF ferritin and S100B in RR-MS group (P=0.004). CSF NOx did not correlate with S100B and ferritin in study groups. RR-MS patients with detectable NfH(SM135) levels had higher NOx compared with subjects having undetectable NfH(SM135) (P=0.03). In the follow-up study, raised baseline levels of NOx (P=0.016) or NfH(SM135) (P=0.04) inversely correlated with the clinical recovery grade expressed as relative EDSS change between baseline and follow-up. In conclusion, NO metabolites were increased and because of their correlation with a biomarker of axonal degeneration (neurofilaments) and a measure for clinical disability (EDSS), relapse-related nitrosative stress is likely to be relevant to the development of sustained disability in an individual patient.

Astrocytic activation in relation to inflammatory markers during clinical exacerbation of relapsing-remitting multiple sclerosis.

The study aimed to assay the cerebrospinal fluid (CSF) levels of protein S100B, a biomarker of astrocyte activation in relation to kynurenic acid (KYNA) and nitric oxide (NO) metabolites, nitrate/nitrite (NOx) concentrations in acute relapse multiple sclerosis (MS) patients. Twenty relapsing-remitting MS (RR-MS) patients and 10 controls were enrolled. RR-MS patients were assessed on the expanded disability status scale (EDSS) and underwent lumbar puncture. The CSF KYNA, NOx and S100B levels were significantly higher in RR-MS group compared to controls (p = 0.01, 0.001, 0.04, respectively). There was a significant correlation between CSF S100B and KYNA (p = 0.01) but not NOx (p > 0.05) in RR-MS. CSF KYNA, NOx or S100B concentrations did not correlate with disease characteristics of MS patients. Our study suggests the activation of the kynurenine pathway leading to the increase of neuroprotective KYNA in the CSF of MS patients during acute relapse what contrasts with chronic phases of the disease.

Simvastatin could prevent increase of the serum MMP-9/TIMP-1 ratio in acute ischaemic stroke.

MMP-9 plays an important role in the pathogenesis of AIS and predicts haemorrhagic transformation of the ischaemic focus. The aim of our study was to analyse both serum MMP-9 and its most specific endogenous inhibitor (TIMP-1) levels in AIS and to check whether HMG-CoA reductase inhibitor (simvastatin) affects the MMP-9/TIMP-1 ratio value. Fifty patients with AIS were randomly divided into two groups: Group I (N = 25) treated with 40 mg/day with simvastatin within 24 hours after the onset of stroke and Group II (N = 25) non-treated with statin. To evaluate MMP-9 and TIMP-1 serum levels, the ELISA method was used. The serum MMP-9 level was significantly elevated on the 7th day of stroke in both groups (from 668 to 862 ng/ml and 670 to 855 ng/ml, respectively, in Group I and II). The serum TIMP-1 level was also elevated on the 7th day of stroke in both groups but the results were not significant. The MMP-9/TIMP-1 ratio was elevated on the 7th day of stroke in both groups, but the result was significant only in the Group II (P < 0.01). These findings indicate that simvastatin given during 24 hours after the onset of stroke could have an influence on the MMP-9/TIMP-1 ratio during AIS.

Increased urinary excretion of nitric oxide metabolites in longitudinally monitored migraine patients.

This study evaluated a relationship between nitric oxide (NO) and migraine attacks in order to gain insight into migraine pathomechanism. The study groups consisted of 12 migraineurs and eight controls. All subjects collected morning urine samples for 40 consecutive days. Urinary NO metabolites, nitrite/nitrate (NO(x)) levels were measured with the vanadium-based assay, whilst creatinine (Cr) and neopterin were determined with high-performance liquid chromatography. The mean urinary NO(x)/Cr ratio and number of NO(x) peaks was significantly greater in the migraine group compared with controls (P = 0.01 and P = 0.007, respectively). In the second approach, high NO(x) values were re-assessed in relation to raised neopterin, a marker of systemic infection or inflammation, and were excluded. The excretion of NO(x) persisted being pulsatile, and migraineurs had more peaks compared with controls (P = 0.01). In seven patients, NO(x) peaks coincided with headache days. This was more frequent than expected by random association in four patients (Monte-Carlo simulation; odds ratios: 2.16-7.77; no overlap of 95% CI). In four patients, NO(x) peaks preceded or followed headache days. Although there is a difference in the pattern of urinary NO(x) excretion between control and migraine populations, the variable temporal association of NO(x) peaks and headaches suggests a complex role of NO in this condition.

The CSF levels of total-tau and phosphotau in patients with relapsing-remitting multiple sclerosis.

Total-tau protein is considered the marker of axon damage whereas the abnormally phosphorylated tau forms are mainly associated with Alzheimer's disease. An increase in total-tau levels was observed in neurodegenerative diseases, including multiple sclerosis (MS). In order to find out whether the phosphorylated tau forms occur in MS patients and to evaluate their clinical significance, the levels of total-tau (t-tau) and tau phosphorylated at Thr 181 (p-tau) were determined in 60 MS patients (40 during relapse including 18 with the first relapse and 20 stable) and in 18 age-matched controls. The determinations were conducted in the cerebrospinal fluid (CSF) using the ELISA method. The levels of t-tau and p-tau were higher in MS patients than in controls; however, increased levels were not related to the clinical activity of the disease. In CSF of the patients with the first relapse the level of t-tau was significantly increased whilst the level of p-tau was not elevated.

Correlations between IL-4, IL-12 levels and CCL2, CCL5 levels in serum and cerebrospinal fluid of multiple sclerosis patients.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS). Both cytokines and chemokines have been implicated in the pathogenesis of MS. The aim of the study was to assess whether cytokine levels are correlated with chemokine levels during a different stage of relapsing-remitting MS (RR-MS). The study included 53 patients with RR-MS (20 subjects in stable stage and 18 patients with relapse). By ELISA method, the levels of the interleukin-4 (IL-4), interleukin-12 (IL-12), CCL2 and CCL-5 chemokines were measured both in serum and cerebrospinal fluid (CSF) of all patients. The serum IL-4 and IL-12 levels and CSF CCL5 level of patients with stable RR-MS were significantly different from the control level and the IL-12 levels were correlated with CCL5 levels in serum. During the relapse, a significant change in chemokine levels both in serum and CSF and IL-12 in CSF were noted, however no correlations were found between cytokines and chemokines.

The levels of chemokines CXCL8, CCL2 and CCL5 in multiple sclerosis patients are linked to the activity of the disease.

Chemokines are small cytokines with selective chemoattractant properties. They contribute to the T-cell-mediated pathogenesis of multiple sclerosis (MS). In order to ascertain whether different types and stage of disease correlate with a varying level of chemokines, the levels of CXCL8, CCL2 and CCL5 were measured in serum and the cerebrospinal fluid (CSF) of the MS patients. ELISA method was used to examine 56 patients with different types of MS alongside the 29 patients of the control group. The levels of CXCL8 and CCL2 in both groups were higher in CFS than in serum whilst the level of CCL5 measured higher in serum than in CSF. A significant rise in the levels of CXCL8 and CCL5 was observed during relapse, as against the level of CCL2 which was lower when compared with the control and other MS groups. No significant differences were observed in the levels of chemokines between the stable relapsing-remitting MS and progressive MS. The different levels of chemokines are linked to relapse of the disease. No separate, specific pattern of chemokine production dependent on the type of MS could be ascertained.

CSF nitric oxide metabolites are associated with activity and progression of multiple sclerosis.

OBJECTIVE: To investigate the relationship of CSF and the serum nitric oxide metabolites nitrite and nitrate (NOx) to disease activity and progression in patients with multiple sclerosis (MS). METHODS: The study was divided into cross-sectional and follow-up. In the cross-sectional study, 20 patients with relapsing-remitting (RR), 21 with secondary progressive (SP), and 10 with primary progressive (PP) MS and 14 control subjects were included. Patients were assessed on clinical (Expanded Disability Status Scale [EDSS], Ambulation Index [AI], 9-Hole Peg Test [9-HPT]) and MRI measurements. In the follow-up study, 34 MS patients from the cross-sectional study agreed to be assessed again after an average of 3.0 +/- 0.5 years. NOx was measured using a vanadium-based assay. RESULTS: In the cross-sectional study, CSF NOx was raised in patients with RR-MS (p = 0.001) and PP-MS (p = 0.02) vs controls. Higher CSF NOx levels were found in patients with mild disability (AI < or = 6.0; EDSS < or = 4.0; Multiple Sclerosis Severity Score [MSSS] < or = 4.8) vs patients with advanced disease (AI > 6.0 [p = 0.002]; EDSS > 4.0 [p = 0.02]; MSSS > 4.8 [p = 0.01]). In the subgroup of patients having Gd-enhancing MRI lesions (n = 11), correlation between the volume of enhancement and CSF NOx was found (r = 0.74, p = 0.01). In the follow-up study, patients with disability progression had higher baseline CSF NOx levels than those who were stable on EDSS (p = 0.02) or AI (p = 0.03). A positive correlation was found between baseline CSF NOx and the change in MR T2-weighted lesion load (r = 0.4, p = 0.03). CONCLUSIONS: CSF nitrite and nitrate levels were increased in mildly disabled patients with MS and found to correlate with the volume of Gd-enhanced lesions on MRI. Raised baseline CSF NOx was associated with clinical and MRI progression in MS patients over 3-year follow-up.

Interleukin-8 and RANTES levels in patients with relapsing-remitting multiple sclerosis (RR-MS) treated with cladribine.

OBJECTIVE: Chemokines are involved in the pathogenesis of multiple sclerosis. The aim of the study was to evaluate the effects of immunosuppressive therapy on production of two proinflammatory chemokines--interleukin-8 (IL-8) and RANTES (regulated on activation, normal T cell expressed and secreted). MATERIALS AND METHODS: Twenty-five patients with relapsing-remitting multiple sclerosis were treated with 2-chlorodeoxyadenosine (Cladribine), administered subcutaneously in 6 cycles repeated every 5 weeks. IL-8 and RANTES levels were measured by the enzyme-linked immunoassay (ELISA) method in serum and cerebrospinal fluid (CSF) before and after treatment. RESULTS: After Cladribine treatment the levels of IL-8 decreased significantly in CSF only, whereas the RANTES levels decreased significantly both in CSF and serum. CONCLUSION: Our results suggest that Cladribine therapy might modify the circulating level of RANTES.

Markers of inflammation in cerebral ischemia.

The study sought to determine whether cerebral ischemia is associated with inflammatory reactions indicated by an increase in levels of selected acute phase proteins (APP), C-reactive protein (CRP), fibrinogen, alpha-1 antitrypsin (AAT) and acidic alpha-1 glycoprotein (AGP). These proteins are thought to be markers of inflammatory reactions. We investigated 30 patients with acute cerebrovascular ischemia, 20 patients with transient ischemic attack, and 20 patients from a control group. Levels of CRP, AAT, AGP, and fibrinogen in blood sera were determined in all patients by kinetic turbidimetry. In the patients with cerebral infarct an increase was found in the levels of APP, which suggests that ischemic necrosis is associated with inflammatory reactions. All patients require active treatment of an inflammatory process that is associated with stroke.