Search

Background: Progressive multifocal leukoencephalopathy (PML) caused by the JC virus is the main limitation to the use of disease modifying therapies for treatment of multiple sclerosis (MS)., Methods: To assess the PML risk in course of ocrelizumab, urine and blood samples were collected from 42 MS patients at baseline (T0), at 6 (T2) and 12 months (T4) from the beginning of therapy. After JCPyV-DNA extraction, a quantitative-PCR (Q-PCR) was performed. Moreover, assessment of JCV-serostatus was obtained and arrangements' analysis of non-coding control region (NCCR) and of viral capsid protein 1 (VP1) was carried out., Results: Q-PCR revealed JCPyV-DNA in urine at all selected time points, while JCPyV-DNA was detected in plasma at T4. From T0 to T4, JC viral load in urine was detected, increased in two logarithms and, significantly higher, compared to viremia. NCCR from urine was archetypal. Plasmatic NCCR displayed deletion, duplication, and point mutations. VP1 showed the S269F substitution involving the receptor-binding region. Anti-JCV index and IgM titer were found to statistically decrease during ocrelizumab treatment., Conclusions: Ocrelizumab in JCPyV-DNA positive patients is safe and did not determine PML cases. Combined monitoring of ocrelizumab's effects on JCPyV pathogenicity and on host immunity might offer a complete insight towards predicting PML risk.

Background: Diet is a modifiable behavior of interest in multiple sclerosis (MS); however, measures of diet in persons with MS have not been vetted for feasibility, acceptability, and validity., Methods: This cross-sectional study examined the Automated Self-Administered 24-H (ASA24) Dietary Assessment Tool in 30 persons with MS and 15 healthy control (HC) participants. Participants were prompted to complete six ASA24 recalls and undergo a standard doubly labeled water (DLW) protocol. Acceptability of ASA24 was assessed using an online questionnaire. Total energy expenditure (TEE) from DLW was compared with ASA24-reported energy intake for assessing validity., Results: All participants completed four or more ASA24 recalls, indicating feasibility of ASA24. Regarding acceptability, the hardest part of completing the ASA24 was remembering everything eaten the previous day. Pearson correlation coefficients between DLW TEE and ASA24 kcal/day were not significant among HC (r = 0.40; p = 0.14) or MS (r = 0.26; p = 0.16) participants. The absolute mean error between DLW TEE and ASA24 among HC participants was 694.96 ± 506.25 mean kcal/day and among MS participants was 585.37 ± 529.02 mean kcal/day; this represents a mean difference of 30 and 25%, respectively., Conclusion: This study established the feasibility and acceptability of ASA24 in persons with MS and provides a foundation regarding the need for further validation research examining appropriate outcomes for supporting dietary interventions.

Background: There is limited information regarding the daily shedding of JC virus (JCV) in urine and its correlation with serum JCV antibody levels., Methods: The dynamic expression of JCV in urine and its correlation with JCV antibody status in patients receiving disease modifying therapy for multiple sclerosis were examined in a longitudinal case-control study. JCV antibody index levels were determined using a two-step ELISA (Stratify). JCV shedding in urine samples was determined by quantitative PCR during two 30-day study periods separated by intervals of at least 6 months., Results: Of 42 study subjects (57% female; ages 22-56, average age 39.6 years), 27 (64.3%) were JCV antibody positive (index >0.40) at initial urine collection. Twelve seropositive subjects (44.4%) had detectable JCV in their urine with values ranging from 290 to 5.08 × 10 8 copies/mL. Daily viral shedding in these patients remained fairly constant throughout the study. Urinary JCV shedding was not detected in any JCV antibody index negative or indeterminate subject. In JCV urinary shedders, the average JCV antibody index was 2.69 (range 1.67-3.57). The average anti-JCV antibody index for the remaining JCV seropositive individuals without viral urinary shedding was 1.35 (range 0.46-3.91)., Conclusion: MS patients displayed a consistent pattern of JCV shedding over days and months in which higher levels of viruria appeared to have driven higher levels of JCV antibody index. The findings provide additional insights into the dynamic expression of JCV and host response; however, studies in larger populations and of longer duration will be needed to determine their significance to the development of progressive multifocal leukoencephalopathy (PML)., Competing Interests: Declaration of Competing Interest Dr. Joseph R. Berger has been a consultant for Amgen, Biogen, Celgene, Genentech/Roche, Merck/Serono, Millennium/Takeda, Novartis, and Shire during the course of the study. He serves on the scientific advisory boards of Excision-Bio and Inhibikase and has received a grant from Biogen for the conduct of this study. Dr. Robert J. Danaher has nothing to disclose. Jessica Dobbins has nothing to disclose. Dr. David Do has nothing to disclose. Dr. Craig S. Miller has nothing to disclose., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) that is associated with demyelination and neuronal loss. Over recent years, the immunological and neuronal effects of tryptophan (Trp) metabolites have been largely investigated, leading to the hypothesis that these compounds and the related enzymes are possibly involved in the pathophysiology of MS. Specifically, the kynurenine pathway of Trp metabolism is responsible for the synthesis of intermediate products with potential immunological and neuronal effects. More recently, Trp metabolites, originating also from the host microbiome, have been identified in MS, and it has been shown that they are differently regulated in MS patients. Here, we sought to discuss whether, in MS patients, a specific urinary signature of host/microbiome Trp metabolism can be potentially identified so as to select novel biomarkers and guide toward the identification of specific metabolic pathways as drug targets in MS., (Copyright © 2020 Gaetani, Boscaro, Pieraccini, Calabresi, Romani, Di Filippo and Zelante.)

Aims: Urodynamics (UDS) is often indicated for multiple sclerosis (MS) patients either at presentation to specialized medical centers or after failure of conservative management of lower urinary tract dysfunction (LUTD). However, the ideal moment and context to indicate this exam in this group of patients remain controversial. We aimed to establish a consensus panel to address the role of UDS in MS patients., Methods: A panel representing urology, rehabilitation medicine, and neurology skilled in neuro-urology participated in a consensus-forming project using a Delphi method to reach consensus on the role of UDS in MS patients., Results: In total, five experts participated. All panelists participated in the four phases of the consensus process. Consensus was reached if ≥70% of the experts agreed on recommendations. To facilitate a common understanding among all experts, a face-to-face consensus meeting was held in Florence in September 2017 and then with a follow-up teleconference in March 2018. By the end of the Delphi process, formal consensus was achieved for 100% of the items and an algorithm was then developed in a face-to-face meeting in Philadelphia in August 2018. The final expert opinion recommendations were approved by the unanimous consensus of the panel., Conclusions: UDS represents an important diagnostic tool for MS patients and is particularly useful to evaluate the pattern of LUT dysfunction in high-risk patients. There is a lack of high-evidence level studies to support an optimal urodynamic long-term follow-up protocol., (© 2019 Wiley Periodicals, Inc.)

Background: Systemic inflammation is a marker of ill health and has prognostic implications in multiple health settings. Urinary neopterin is an excellent candidate as a nonspecific marker of systemic inflammation. Expression as urinary neopterin-to-creatinine ratio (UNCR) normalizes for urinary hydration status. Major attractions include (a) urine vs blood sampling, (b) integration of inflammation over a longer period compared with serum sampling, and (c) high stability of neopterin and creatinine., Methods: A high-throughput ultraperformance LC-MS method was developed to measure neopterin and creatinine together from the same urine sample. The assay was applied in several clinical scenarios: healthy controls, symptomatic infections, and multiple sclerosis. Area under the curve was compared between weekly and monthly sampling scenarios. Analysis of a single pooled sample was compared with averaging results from analysis of individual samples., Results: The assay has excellent intraassay and interassay precision, linearity of dilution, and spike and recovery. Higher UNCR was demonstrated in female vs male individuals, older age, inflammatory disease (multiple sclerosis), and symptomatic infections. In healthy controls, fluctuations in inflammatory state also occurred in the absence of symptomatic infection or other inflammatory triggers. Analysis of a single pooled sample, made up from weekly urine samples, integrates inflammatory activity over time., Conclusions: UNCR is a useful biomarker of systemic inflammation. The method presented offers simplicity, speed, robustness, reproducibility, efficiency, and proven utility in clinical scenarios. UNCR fluctuations underline the importance of longitudinal monitoring, vs a single time point, to capture a more representative estimate of an individual's inflammatory state over time., (© 2019 American Association for Clinical Chemistry.)

Identification of non-invasive biomarkers of disease progression in multiple sclerosis (MS) is critically needed for monitoring the disease progression and for effective therapeutic interventions. Urine is an attractive source for non-invasive biomarkers because it is easily obtained in the clinic. In search of a urine metabolite signature of progression in chronic experimental autoimmune encephalomyelitis (EAE), we profiled urine at the chronic stage of the disease (day 45 post immunization) by global untargeted metabolomics. Using a combination of high-throughput liquid-and-gas chromatography with mass spectrometry, we found 105 metabolites (P < 0.05) significantly altered at the chronic stage, indicating a robust alteration in the urine metabolite profile during disease. Assessment of altered metabolites against the Kyoto Encyclopedia of Genes and Genomes revealed distinct non-overlapping metabolic pathways and revealed phenylalanine-tyrosine and associated metabolism being the most impacted. Combined with previously performed plasma profiling, eight common metabolites were significantly altered in both of the biofluids. Metaboanalyst analysis of these common metabolites revealed that phenylalanine metabolism and Valine, leucine, and isoleucine biosynthetic pathways are central metabolic pathways in both bio-fluids and could be analyzed further, either for the discovery of therapeutics or biomarker development. Overall, our study suggests that urine and plasma metabolomics may contribute to the identification of a distinct metabolic fingerprint of EAE disease discriminating from the healthy control which may aid in the development of an objective non-invasive monitoring method for progressive autoimmune diseases like MS. Graphical Abstract Untargeted urinary metabolomics of a chronic mouse model of multiple sclerosis identified Phenylalanine, tyrosine & tryptophan metabolism as the significantly altered metabolic pathway. Eight common metabolites were identified when we combined urinary and plasma metabolic signature, which revealed a perturbation of Phenylalanine metabolism and valine, leucine & isoleucine metabolic pathways, involved in CNS dysfunction during diseases. The identified eight metabolic signature of urine and plasma may be of clinical relevance as potential biomarkers and guide towards the identification of specific metabolic pathways as novel drug targets.

Lower urinary tract dysfunction is common among neurological patients. Traditionally, the basic method of diagnosis is a complex urodynamic study. In recent years, many studies have focused on the search for new non-invasive diagnostic modalities. In particular, neurotrophins are considered as potential biological markers of a neurogenic bladder., Aim: To estimate the sensitivity and specificity of the serum and urinary nerve growth factor (NGF) and brain neurotrophic factor (BDNF) in MS patients as markers of detrusor overactivity., Materials and Methods: The study comprised 20 patients with multiple sclerosis, who complained of voiding problems. The control group consisted of 20 people without neurological diseases, lower urinary tract symptoms and detrusor overactivity estimated by filling cystometry. Apart from standard laboratory tests, diagnostic evaluation included a complex urodynamic study, ultrasound of the urinary tract, cystoscopy, testing serum and urinary NGF and BDNF using the enzyme immunoassay. The diagnostic significance of neurotrophins was evaluated using ROC analysis., Results: According to the ROC analysis, the diagnostic sensitivity and specificity of serum NGF as a marker of detrusor hyperactivity was 57% and 93%, respectively (for serum NGF more or equal 26 pg/ml). The quality of the test according to the expert scale of AUC values was "very good" (AUC=0.806). Detecting NGF in patients urine was less effective. The sensitivity and specificity were 52% and 40%, respectively (for NGF more or equal 6 pg/ml). The quality of the test according to the expert scale of AUC values was "average" (AUC=0.64). The serum BDNF demonstrated high sensitivity (90%) and low specificity (23%), AUC=0.56. The urinary BDNF was more informative, (AUC=0.65). The combination of all four markers provides a sensitivity of 85.7% and a specificity of 66.7% (AUC=0.824)., Conclusions: Testing serum and urinary neurotrophins in patients with multiple sclerosis can be used to diagnose detrusor overactivity. The NGF is a highly specific biomarker, while the BDNF is highly sensitive. Combined testing for serum NGF and BDNF is most informative.

Background: Dimethyl fumarate (DMF) is approved for use in patients with relapsing-remitting multiple sclerosis (MS). Its mechanism of action is still not well understood, but besides the immunological pathways in MS, it may also affect the metabolism of normally functioning internal organs, tissues and cells., Case Presentation: We report on the case of 29-year-old woman with satisfactorily-controlled type 1 diabetes (T1D), who was diagnosed as having MS. After administration of DMF she experienced intense, adverse gastro-intestinal reactions together with ketonuria up to 160 mg/dL. The highest ketone concentrations in the urine were observed approximately 2 h after each DMF dose and always with co-existing adverse reactions. Dose reduction did not improve symptoms and treatment had to be stopped. Twelve hours after the last dose of DMF all laboratory results returned to normal ranges and all gastro-intestinal adverse reactions were resolved within the following 24 h., Conclusion: This is a first report of ketonuria in a MS-patient with T1D treated with DMF. Patients with MS and co-existing metabolic diseases, which are not contraindicated for DMF treatment, represent a unique opportunity to address questions regarding the possible mechanisms of action of DMF on the cellular metabolism. The use of DMF in patients with metabolic diseases needs closer attention., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Objective: To compare stone composition and serum/urine biochemistries in stone formers with multiple sclerosis (MS) against stone formers without MS and to examine the association between mobility, methods of bladder emptying, and stone formation., Patients and Methods: In this retrospective case-control study, we identified patients diagnosed with MS and kidney stone disease who were seen at our institution between 2001 and 2016. For the first part of the study, up to two controls (stone formers without a history of MS) were identified for each case and matched on age, body mass index, and sex. For the second part of this study, matched controls (MS patients without a history of stones) were identified in a 1:1 ratio in a similar fashion. Results of 24-h urine biochemistry studies, stone compositions, serum laboratory measures, medications, history of stone surgeries, mobility, and method of bladder emptying were collected., Results: In all, 587 patients were identified who had both MS and a history of stone disease. Of these, 118 patients had a stone composition available. When compared to matched controls, patients with MS were significantly more likely to have calcium phosphate stones (42% vs 15%, P < 0.001) and struvite stones (8% vs 3%, P = 0.03) and less likely to have calcium oxalate monohydrate stones (39% vs 64%, P < 0.001). Among those patients with a composition available, those with MS were more likely to have undergone a percutaneous nephrolithotomy (PCNL; 25% vs 12%, P = 0.005) or a cystolithopaxy (16% vs 3%, P < 0.001) compared to their matched controls. In all, 61 patients had a complete 24-h urinary stone panel. There were no significant differences in urinary pH, volume, creatinine, calcium, citrate, oxalate, sodium, and uric acid as well as rates of hypocitraturia, hyperoxaluria, hypercalciuria, and hyperuricosuria among patients with MS. Use of intermittent straight catheterisation [ISC; odds ratio (OR) 3.50, 95% confidence interval (CI) 1.89-6.47]; P < 0.001] or an indwelling catheter (OR 9.78, 95% CI 4.81-19.88; P < 0.001) for bladder emptying was significantly associated with stone disease. There was no association between level of mobility and stone disease (P = 0.10)., Conclusions: Similar to findings seen in patients with spinal cord injuries, patients with MS have a high incidence of calcium phosphate stones and struvite stones when compared with matched controls. Additionally, they were more likely to undergo PCNL. The method of bladder management appears to be a risk factor in the development of stone disease. These findings suggest the importance of prompt treatment of urinary tract infections in this population and delay the use of ISC, suprapubic tube, or an indwelling Foley, when possible., (© 2017 The Authors BJU International © 2017 BJU International Published by John Wiley & Sons Ltd.)

The nuclear magnetic resonance (NMR)-based metabolomic approach was used as analytical methodology to study the urine samples of chronic inflammatory rheumatic disease (CIRD) patients. The urine samples of CIRD patients were compared to the ones of both healthy subjects and patients with multiple sclerosis (MS), another immuno-mediated disease. Urine samples collected from 39 CIRD patients, 25 healthy subjects, and 26 MS patients were analyzed using 1 H NMR spectroscopy, and the NMR spectra were examined using partial least squares-discriminant analysis (PLS-DA). PLS-DA models were validated by a double cross-validation procedure and randomization tests. Clear discriminations between CIRD patients and healthy controls (average diagnostic accuracy 83.5 ± 1.9%) as well as between CIRD patients and MS patients (diagnostic accuracy 81.1 ± 1.9%) were obtained. Leucine, alanine, 3-hydroxyisobutyric acid, hippuric acid, citric acid, 3-hydroxyisovaleric acid, and creatinine contributed to the discrimination; all of them being in a lower concentration in CIRD patients as compared to controls or to MS patients. The application of NMR metabolomics to study these still poorly understood diseases can be useful to better clarify the pathologic mechanisms; moreover, as a holistic approach, it allowed the detection of, by means of anomalous metabolic traits, the presence of other pathologies or pharmaceutical treatments not directly connected to CIRDs, giving comprehensive information on the general health state of individuals. Graphical abstract NMR-based metabolomic approach as a tool to study urine samples in CIRD patients with respect to MS patients and healthy controls.

Fingolimod is a sphingosine-1-phosphate receptor agonist used to inhibit the inflammatory activity of multiple sclerosis (MS), and has been shown to suppress osteoporosis in mouse models. In this study, levels of bone turnover markers were quantified in serum and urine samples from MS patients treated with fingolimod. Compared with untreated MS patients and healthy controls, fingolimod-treated MS patients had a significantly lower level of the bone resorption marker type I collagen cross-linked N-telopeptide in urine. This finding was prominent in female but was not seen in male subjects. Our results suggest that fingolimod may have a beneficial effect on bone mass loss in female MS patients., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Clinically isolated syndrome (CIS) is a first, usually recovering, episode of neurological disturbance(s) suggestive of multiple sclerosis (MS). CIS subjects might benefit from early disease-modifying drugs, provided that those at high risk of developing MS can be identified. Gene expression for dopaminergic receptors (DR) and adrenoceptors (AR) is dysregulated in lymphocytes of MS patients and is affected by treatment with interferon (IFN)-β. In particular, lymphocyte DR D5 mRNA might be a marker of IFN-β response in MS patients. No information exists so far in CIS subjects. We investigated DR and AR gene expression in peripheral blood mononuclear cells (PBMC) and in CD4+ T effector (Teff) and regulatory (Treg) cells from CIS subjects, and assessed their relationship with MS progression after 12months. Expression of several DR and AR are upregulated in PBMC, Teff and Treg from CIS subjects. DR D3 and α2A-AR mRNA in PBMC, and DR D5 mRNA in Treg correlate with the risk of MS at 12months. Results show the involvement of dopaminergic and adrenergic pathways in CIS as well as in MS pathogenesis, supporting the evaluation of dopaminergic and adrenergic agents in MS., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Urine is a metabolite-rich biofluid that reflects the body's effort to maintain chemical and osmotic homeostasis. Clinical diagnosis routinely relies on urine samples because the collection process is easy and noninvasive. Despite these advantages, urine is an under-investigated source of biomarkers for multiple sclerosis (MS). Nuclear magnetic resonance spectroscopy (NMR) has become a common approach for analyzing urinary metabolites for disease diagnosis and biomarker discovery. For illustration of the potential of urinary metabolites for diagnosing and treating MS patients, and for differentiating between MS and other illnesses, 38 urine samples were collected from healthy controls, MS patients, and neuromyelitis optica-spectrum disorder (NMO-SD) patients and analyzed with NMR, multivariate statistics, one-way ANOVA, and univariate statistics. Urine from MS patients exhibited a statistically distinct metabolic signature from healthy and NMO-SD controls. A total of 27 metabolites were differentially altered in the urine from MS and NMO-SD patients and were associated with synthesis and degradation of ketone bodies, amino acids, propionate and pyruvate metabolism, tricarboxylic acid cycle, and glycolysis. Metabolites altered in urine from MS patients were shown to be related to known pathogenic processes relevant to MS, including alterations in energy and fatty acid metabolism, mitochondrial activity, and the gut microbiota.

Background and Purpose: Progressive multifocal leukoencephalopathy (PML) cases have arisen amongst multiple sclerosis patients treated with natalizumab. Our objective was to gain a better understanding of the mechanisms that underlie the John Cunningham virus (JCV) infection which causes PML., Methods: A study was made of (i) the quarterly JCV DNA levels in peripheral blood mononuclear cells (PBMCs), serum and urine samples in 100 multiple sclerosis patients during their natalizumab treatment (3-39 months), (ii) the association between human leukocyte antigen (HLA) class II and the previous viral detection and (iii) the identification of the JCV variants in those patients suspected of having PML., Results: (i) JCV DNA in PBMCs and/or serum was detected in 23% of our cohort. Patients with an intermittent JCV excretion in urine had a significant increase of the viral load and prevalence in this compartment during natalizumab treatment. (ii) The frequency of the DRB1*07/DQA1*02:01/DQB1*02:02 haplotype tended to be higher in patients with detectable versus undetectable JCV DNA in PBMCs (P(corrected) = 0.108). (iii) The variants in PBMCs and serum of the non-PML patient matched the archetype. In the patient with non-fatal PML, the archetype and the same neurotropic variant in PBMCs, serum and cerebrospinal fluid was identified at the time PML was diagnosed, whereas in the patient with a worse PML prognosis, four neurotropic variants in the three previous compartments were found by the PML diagnosis., Conclusions: The detection of the neurotropic variant in blood during natalizumab treatment could be critical in the prevention of the development of severe PML, since this variant appears simultaneously with the clinical symptoms of PML and mutates quickly., (© 2015 EAN.)

Objectives: Inflammatory demyelinating diseases of the CNS comprise a broad spectrum of diseases like neuromyelitis optica (NMO), NMO spectrum disorders (NMO-SD) and multiple sclerosis (MS). Despite clear classification criteria, differentiation can be difficult. We hypothesized that the urine proteome may differentiate NMO from MS., Methods: The proteins in urine samples from anti-aquaporin 4 (AQP4) seropositive NMO/NMO-SD patients (n = 32), patients with MS (n = 46) and healthy subjects (HS, n = 31) were examined by quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) after trypsin digestion and iTRAQ labelling. Immunoglobulins (Ig) in the urine were validated by nephelometry in an independent cohort (n = 9-10 pr. groups)., Results: The analysis identified a total of 1112 different proteins of which 333 were shared by all 109 subjects. Cluster analysis revealed differences in the urine proteome of NMO/NMO-SD compared to HS and MS. Principal component analysis also suggested that the NMO/NMO-SD proteome profile was useful for classification. Multivariate regression analysis revealed a 3-protein profile for the NMO/NMO-SD versus HS discrimination, a 6-protein profile for NMO/NMO-SD versus MS discrimination and an 11-protein profile for MS versus HS discrimination. All protein panels yielded highly significant ROC curves (AUC in all cases >0.85, p≤0.0002). Nephelometry confirmed the presence of increased Ig-light chains in the urine of patients with NMO/NMO-SD., Conclusion: The urine proteome profile of patients with NMO/NMO-SD is different from MS and HS. This may reflect differences in the pathogenesis of NMO/NMO-SD versus MS and suggests that urine may be a potential source of biomarkers differentiating NMO/NMO-SD from MS.

Multiple sclerosis (MScl) frequently is remitted during the third trimester of pregnancy but exacerbated in the first postpartum period. In this context, we investigated protein identification, its abundance, and its change in urine related to these two periods. Using mass spectrometry (LTQ Orbitrap), we identified 1699 tryptic peptides (related to 402 proteins) in urine from 31 MScl and 8 control at these two periods. Pregnancy-related peptides were significantly elevated (p < 0.01) in MScl patients compared with controls (Analysis 1: 531 peptides in MScl and 36 peptides in controls higher abundant in the third trimester compared to postpartum). When comparing the longitudinal differences (Analysis 2), we identified 43 (related to 35 proteins) MScl disease-associated peptides (p < 0.01) with increased or decreased difference ratio in MScl compared with controls. The most discriminating peptides identified were trefoil factor 3 and lysosomal-associated membrane protein 2. Both proteins have a role in the innate immune system. Three proteins with a significant decreased ratio were plasma glutamate carboxypeptidase, Ig mu chain C region, and osteoclast associated immune like receptor. Our results indicate that the protein expression pattern in urine of MScl patients contains information about remote CNS and brain disease processes.

Lipid peroxidation due to oxidative stress (OS) may play an important role in the pathogenesis of chronic systemic inflammatory diseases such as multiple sclerosis (MS). Telomeres, repeated sequences that cap chromosome ends, undergo shortening with each cycle of cell division, resulting in cellular senescence. Research regarding telomere shortening has provided novel insight into the pathogenesis of various diseases. We hypothesized that OS damage leads to inflammatory reactions, which subsequently shortens the telomere length in MS. We enrolled 59 patients with MS, and age- and gender-matched 60 healthy controls. We divided MS subjects into three groups matched for age and gender according to the severity of disability: relatively benign course (BMS), secondary progressive MS, and primary progressive MS (PPMS). We analyzed the telomere length in peripheral blood mononuclear cells and the 8-iso-PGF2α concentration in urine, a reliable and stable marker of lipid peroxidation in vivo. The data showed significant higher levels of urinary 8-iso-PGF2α in MS subjects than in the controls. The lag-time, which represents the direct measurement of the resistance of low-density lipoprotein to oxidation, was shorter in the PPMS subjects than in the groups. Compared to that observed in the controls, the mean telomere length was significantly shorter in the PPMS group, whereas no significant telomere shortening was found between the controls and other subjects. Our data suggest that a decreased telomere length and enhanced lipid peroxidation reflects the severest stage of MS.

Objectives: Biomarkers with the potential for longitudinal measurements are needed in multiple sclerosis (MS). Urine is easy to collect, and repeated sampling is possible., Methods: 39 paired CSF and urine samples were taken. Oligoclonal bands (OCBs) were measured in CSF. Kappa and lambda free light chain (FLC), neopterin and ubiquitin C-terminal hydrolase-L1 (UCHL1) were measured in CSF and urine., Results: 16/39 samples had OCBs unique to the CSF. CSF FLC levels (P < 0.0001) were higher in OCB-positive subjects, with no difference in urinary FLC. CSF and urinary FLC did not correlate. There were a significant correlation between total CSF FLC and CSF neopterin in MS samples (correlation coefficient = 0.588, P = 0.016) and a strong correlation between CSF lambda FLC and CSF neopterin in MS samples (correlation coefficient = 0.875, P < 0.001). There was a strong correlation between urinary neopterin/creatinine levels and urinary total FLC/protein levels (correlation coefficient = 0.452, P = 0.004). Only three CSF samples (8%) had detectable levels of UCHL1. 18/38 (48%) (8/15 MS and 10/23 control) urine samples had detectable levels of UCLH1., Conclusions: This study confirms the relationship between CSF OCBs and CSF FLCs, highlighting the importance of intrathecal B- and plasma-cell activation in MS. There is a relationship between CSF FLC and CSF neopterin in MS, highlighting the multifaceted immune activation seen in MS. Correlations in the OCB-positive group highlight the multifaceted immune activation seen in MS. Further studies are required to evaluate CSF and urinary biomarkers., (© 2013 John Wiley & Sons A/S.)

Natalizumab-associated progressive multifocal leukoencephalopathy in multiple sclerosis (MS) occurred in two individuals also treated with interferon β1a, raising concerns about the interaction of these disease-modifying agents and leading to the recommendation to avoid their concomitant administration. However, type I interferons are antiviral. Using a real-time quantitative polymerase chain reaction for the detection and quantification of the John Cunningham virus (JCV), DNA in peripheral blood mononuclear cells (PBMCs), and urine in MS patients, we tested the hypothesis that MS disease-modifying drugs (DMD) qualitatively and quantitatively alter JCV prevalence and viral copy numbers. Two hundred thirty-nine patients were enrolled in a cross-sectional study in which blood and urine specimens were collected at a single time and 37 newly diagnosed, treatment-naïve MS patients were enrolled in a longitudinal study in which specimens were obtained at diagnosis and 6 months after treatment initiation. JCV DNA was detected in PBMCs of only two patients (0.07 %), but was commonly detected in the urine (46.8 %) in this population. There was no effect of DMDs on blood or urinary JCV prevalence or viral copy numbers with either glatiramer acetate (Copaxone®) or interferon-β therapy (Avonex®, Betaseron®, or Rebif®). The small number of patients on other therapies precluded meaningful comment about their effects. No obvious effect of the platform DMDs on JCV prevalence was observed even for the interferon-βs.

Objective: A study was undertaken to define the prevalence of anti-JC virus (JCV) antibodies in multiple sclerosis (MS) patients and to evaluate the analytical false-negative rate of a 2-step anti-JC virus antibody assay., Methods: STRATIFY-1 is an ongoing, longitudinal, observational study of relapsing MS patients in the United States who are being treated or considering treatment with natalizumab. Baseline serum and plasma samples were collected for anti-JC virus antibody detection using an analytically validated, 2-step, virus-like particle-based enzyme-linked immunosorbent assay. Urine was collected for JC virus DNA detection., Results: At baseline (n = 1,096), overall anti-JC virus antibody prevalence was 56.0% (95% confidence interval [CI], 53.0-59.0) in STRATIFY-1 patients, with an assay false-negative rate of 2.7% (95% CI, 0.9-6.2). Prevalence was significantly lower in females (53.4%; 95% CI, 49.9-56.8) than males (64.3%; 95% CI, 58.2-70.0) and increased with age, p = 0.0019 and p = 0.0001, respectively. Prevalence was similar in patients regardless of natalizumab exposure or prior immunosuppressant use, p = 0.9709 and p = 0.6632, respectively. STRATIFY-1 results were generally consistent with those observed in another large North American cohort, TYGRIS-US (n = 1,480)., Interpretation: Baseline results from STRATIFY-1 are consistent with other studies utilizing this assay that demonstrate a 50 to 60% prevalence of anti-JC virus antibodies, a low false-negative rate, and an association of increasing age and male gender with increasing anti-JC virus antibody prevalence. Neither natalizumab exposure nor prior immunosuppressant use appear to affect prevalence. Longitudinal data from STRATIFY-1 will confirm the stability of anti-JC virus antibody prevalence over time., (Copyright © 2011 American Neurological Association.)

Objective: To characterize the effect of vitamin D(3) intake on urinary calcium:creatinine ratios across predefined ranges of serum 25(OH)D., Design: Patients with multiple sclerosis (n=25) received escalating doses of vitamin D(3) (4000-40,000IU/d) with calcium (1200mg/d)., Results: Urinary calcium:creatinine was driven by increased 25(OH)D when concentrations were <75nmol/L (r=0.424, p=0.009) and >200nmol/L (r=0.281, p=0.01), but no relationship existed when 25(OH)D concentrations were 76-200nmol/L., Conclusions: A "safe", physiological range of 25(OH)D concentrations is 75-200nmol/L., (Copyright © 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Bone mineral density (BMD) is affected in young adults with multiple sclerosis (MS), which leads to disabling disease. We aimed to show changes that were independent of immobilization by measuring BMD and laboratory markers of bone metabolism in mobile MS patients. We compared a total of 52 premenopausal female patients with relapsing-remitting multiple sclerosis (RRMS) to 41 women of similar age who had no risk factors for osteoporosis. The lumbar and femur BMD were measured using the dual energy X-ray absorptiometry (DXA) method. The urine concentration of serum 25-hydroxycholecalciferol (25-OH vit D(3)), and pyridinoline and deoxypyridinoline were also measured. The concentration of serum osteocalcin was measured to determine the speed of bone metabolism. The mean age of patients (+/- standard deviation [SD]) was 36.1+/-7.4. The average Expanded Disability Status Scale (EDSS) score was 2.2+/-1.8. The concentration of 25-OH vit D(3) and osteocalcin was lower, whereas the concentration of parathyroid hormone (PTH), alkaline phosphatase (ALP), pyridinoline and deoxypyridinoline was higher in the patient group. In the patient group, lumbar 2-4 BMD, T score and Z score and femur neck and trochantor BMD, T score and Z score were significantly lower than in the control group. There was a significant negative relationship between: the disease period and L 2-4 BMD, T score and Z scores; and the femoral neck BMD, T score and Z scores. There was a significant relationship between the total Functional Independence Measure score and the femoral neck, femoral trochanter BMD, T score, and Z score. There was a significant negative relationship between the average EDSS, L 2-4 and all the DXA measurements obtained from the femur. There was a significant relationship between the 25-OH vit D(3) concentration and L 2-4 T score and Z score from the DXA measurements obtained from the femur. There were no significant relationships between osteocalcin, pyridinoline, deoxypyridinoline levels and the BMD measurements. Therefore, the duration of the disease and decrease in functional capacity are the main factors that affect BMD in MS. Apart from the decrease in functional capacity, 25-OH vit D(3) deficiency and secondary PTH increase contribute to the BDM changes observed in MS., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Background: Plasma and B cells are implicated in multiple sclerosis (MS) and produce free light chains (FLC) that are excreted in urine., Objective: To confirm that demyelinating diseases (DD) cause increased urinary FLCs., Method: Urinary FLC in 50 patients with DD were compared to 20 patients with posterior uveitis (PU), 19 with AIDS, 34 with rheumatoid arthritis (RA) and 19 normal controls (NC)., Result: Subjects with DD, PU, RA and AIDS have higher urinary FLCs than NC (p<0.01). Urinary FLCs did not correlate with gadolinium-enhancing lesions on MRI., Conclusions: Urinary FLCs are raised in DD. Further studies are required to see if they correlate with disease activity., (Copyright 2010 Elsevier B.V. All rights reserved.)

Aim: To determine whether patients complaining of oral and medical symptoms perceived to be associated with chronic mercury toxicity have elevated mercury levels in their blood and urine., Methods: The study group in this audit were 56 patients presenting to an oral medicine unit with complaints perceived to be related to chronic mercury toxicity. Their symptoms and co-morbidity were charted and mercury levels in blood and urine were biochemically tested by atomic absorption spectrophotometry., Results: None had elevated mercury levels in blood or urine above the normal threshold level. Subgroup analysis showed subjects with oral lesions, autoimmune disorders and multiple sclerosis had relatively and significantly higher mercury levels within this cohort, but within the threshold values. When tested by multiple logistic regression adjusted for age and gender, mercury levels in blood or urine, numbers of amalgams were not significant for multiple sclerosis or previously diagnosed autoimmune disease., Conclusion: Mercury levels in blood and urine of this cohort of patients with perceived chronic mercury toxicity were within the normal range in accordance with a national laboratory threshold value.

Objective: The purpose of this study was to examine the circadian distribution of creatinine and uric acid clearances in subjects with Multiple Sclerosis., Materials and Methods: Eleven subjects with MS, 6 women (48+/-7y) and 5 men (58+/-5y) volunteered for this circadian study. Thirteen healthy females (39+/-11y) served as controls. Data of seven healthy male controls (64+/-8 y) were extracted from a similar circadian study conducted previously. Each MS patient, and each male control had blood samples drawn around the clock, at 3h intervals (8/24h), and each collected urines over 3h periods (8/24h). Each female control contributed only one blood sample and one complete 24h urine collection. Blood and urine samples were analyzed for a number of relevant analytes: ELAM, IL-6, NO, insulin, ACTH, aldosterone, cortisol, electrolytes, lymphocytes, monocytes including creatinine and uric acid clearances. Those were standardized to an average body surface area of 1.73 m2., Results: The relevant analytes demonstrated increased synthesis of insulin, IL-6, ELAM, monocytes, and reduced concentrations of serum NO. The creatinine clearances were significantly lower in MS females than in female controls, 63+/-22 vs.108+/-18 ml/min. They were also lower than those of MS males and male controls, 107.8+/-17, 97.5+/-8.2 ml/min. Uric acid clearances in MS females were also lower 6.9+/-2.4 vs. 10.5+/-4.4 ml/min. The uric acid clearance in MS males was higher than in male controls, 7.0+/-4.5 vs. 4.0+/-1.0 ml/min., Conclusions: The alterations in selected relevant analytes and the reduced creatinine and uric acid clearances in females but not in males, suggest a renal dysfunction in MS females. These observations may contribute to understanding better the mechanism of renal dysfunction in female patients and perhaps this may be an additional factor contributing to greater frequency of MS in females than in male subjects.

Multiple sclerosis (MS) is a chronic, immune-mediated, demyelinating disease of the central nervous system of as yet unknown aetiology. A consensus of opinion has suggested that the disorder is the result of an interplay between environmental factors and susceptibility genes. We have used a battery of analytical techniques to determine if the urinary excretion of i) markers of oxidative damage; ii) iron and iii) the environmental toxin aluminium and its antagonist, silicon, are altered in relapsing-remitting (RRMS) and secondary progressive MS (SPMS). Urinary concentrations of oxidative biomarkers, MDA and TBARS, were not found to be useful indicators of inflammatory disease in MS. However, urinary concentrations of another potential marker for inflammation and oxidative stress, iron, were significantly increased in SPMS (P<0.01) and insignificantly increased in RRMS (P>0.05). Urinary concentrations of aluminium were also significantly increased in RRMS (P<0.001) and SPMS (P <0.05) such that the levels of aluminium excretion in the former were similar to those observed in individuals undergoing metal chelation therapy. The excretion of silicon was lower in MS and significantly so in SPMS (P<0.05). Increased excretion of iron in urine supported a role for iron dysmetabolism in MS. Levels of urinary aluminium excretion similar to those seen in aluminium intoxication suggested that aluminium may be a hitherto unrecognized environmental factor associated with the aetiology of MS. If aluminium is involved in MS then an increased dietary intake of its natural antagonist, silicon, might be a therapeutic option.

Objective: To investigate circadian rhythm (CR) of urinary creatinine and 8-hydroxy-2-deoxyguanosine (8-OHdG) in patients with Multiple Sclerosis (MS) and to present concentrations of this DNA damage marker, 5 years prior to mastectomy, in one MS study subject, and 2 years prior to biopsy confirmed a carcinoma (CA) of the prostate in one non-MS subject., Materials and Methods: Eleven subjects with MS (6 women 36-52 years of age and 5 men 51-68 years) volunteered for this study, carried out at Edward Hines Jr., Medical Center. Subjects were offered a general hospital diet (2400 cal in total/24h) at 16:30h, 07:30h and 13:00h. The dark (sleep) phase of the light-dark cycle extended from 22:30h to 06:30h with brief awakening for sampling at 01:00h, and 04:00h. Urine samples were collected for consecutive 3h spans beginning at 16:00-19:00h and were analyzed for creatinine and 8-OHdG. Twelve men (including 3 with type 2 diabetes) provided 21 profiles according to the same protocol used for comparison. In addition, 10 healthy women provided 24h urine samples. Statistical analysis of data was performed using the Single-Cosinor and Population-Mean Cosinor., Results: A CR was detected for creatinine in healthy men (p < 0.001) but not for MS patients. Urinary creatinine concentrations were lower in MS women than in healthy women (p = 0.015) and were lower in MS women than in men healthy or with MS (p < 0.001): Women; MS 655 +/- 76; H 1381 +/- 316; Men, MS 1830 +/- 285; H 1532 +/- 265 mg/24h vol. A CR was evident in 8-OHdG in MS (p = 0.007) and in non-MS subjects (p < 0.001) with highest values occurring at about 16:45h. The average concentrations of 8-OHdG in MS patients were similar to those in healthy subjects: Women, MS 589 +/- 125; H 794 +/- 318; Men, MS 504 +/- 156; H 591 +/- 134 picomoles/kg bw/24h vol. The 8-OHdG concentrations of a MS patient, later diagnosed with breast cancer, were found to exceed the upper 95% prediction limit in health. An increased 8-OHdG level was also noted in a non-MS subject who 2 years later received a biopsy-confirmed diagnosis of prostate CA., Conclusions: Despite the small number of subjects in this study, a statistically significant CR was documented for 8-OHdG in urine of subjects with MS. Interestingly, the increased concentrations of DNA damage marker, the 8-OHdG, 5 years prior to mastectomy and the 2 years prior to affirmative diagnosis of prostate CA, could be the most significant clinical observations of this study. Follow-up studies of a larger population of subjects would, thus, be required to ascertain the predictive validity of such challenging observation.

Muliple sclerosis (MS) is a chronic disorder of autoimmune origin, affecting young people. The aim of our study was to evaluate the tobacco smoking in the patients with MS in the period of beginning the disease. The study included 58 MS patients (31 females, 27 males, mean +/- SD: age 32.15 +/- 6.28) with varying degrees of severity and types of MS (A. RRMS relapsing- remitting MS, B. SPMS - secondary-progressive MS and C. PPMS primary - progressive MS). Smoking status was determined by medical history and Fagerström test. In the examined group 38 (65.51%) persons were tobacco smokers, among them 21 (55.26%) females and 17 (44.74%) males. This preliminary analysis needs further thorough studies, f.e. the urine level of cotinine - as a main marker of tobacco smoking in MS patients.

DNA-hydrolyzing antibodies (DNA-abzymes, Abz) were shown to be good biochemical markers of some autoimmune diseases such as systemic lupus erythematosus (SLE) and multiple sclerosis (MS). To better understand mechanisms of abzyme generation, one needs to know optimal conditions for DNA hydrolysis by DNA-abzymes, as well as their enzymatic properties in comparison with those of enzymes possessing the same activity. In contrast to human urine deoxyribonucleases, DNA-hydrolyzing antibodies efficiently digested both single- and double-strand DNA. It was shown that polyclonal antibodies (Abs) in MS may contain up to several types of DNase activities, either activated by metal ions or not.

Multiple sclerosis (MS) is a predominately T helper 1-mediated autoimmune disease of the central nervous system. Magnetic resonance imaging (MRI) is the most useful tool for monitoring disease activity and progression. However, MRI is an expensive and time-consuming test. Therefore, the ability to measure biological markers in body fluids correlating with MRI disease activity is of great importance. Beta-2 microglobulin (beta2-MG) and neopterin have been found to correlate with disease activity in several autoimmune disorders and are used as pharmacodynamic markers of interferon beta treatment in MS. During the natural course of MS, beta2-MG is stable over time, and thus it is unlikely that monitoring its plasma levels will be a useful marker of disease changes. More controversial results have been found for neopterin evaluations in MS. Urinary excretion of neopterin is higher during a clinical relapse but blood levels of this molecule do not correlate with clinical and MRI measurements.

Proton nuclear magnetic resonance (1H-NMR) spectroscopy in combination with pattern recognition techniques were used to investigate the composition of organic compounds in urines from patients with multiple sclerosis (MS), patients with other neurological diseases (OND) and healthy controls (H). Using a valid animal model of MS, namely the common marmoset (Callithrix jacchus) model of experimental autoimmune encephalomyelitis (EAE), the relation of disease progression and alteration of the urine composition was investigated. Urine samples were collected during different stages of EAE, either induced with whole human myelin or with the myelin protein MOG in complete adjuvant. The urine samples were analysed with 1H-NMR spectroscopy allowing simultaneous detection of an array of compounds. Spectral differences between urines from EAE-affected and healthy monkeys were assessed with multivariate analysis. Evidence is provided that development of EAE is associated with changes in the chemical composition of the urine, in particular of compounds with NMR peaks in the region of the spectrum between 0.5 and 3.50 ppm. In addition, we found preliminary evidence for differences between urines from MS, OND and H groups.

Objectives: The role of leukotrienes (LTs) in the pathophysiology of multiple sclerosis (MS) has been controversially discussed in the past. Studies of LTs in the cerebrospinal fluid (CSF) revealed different results mainly because of analytical difficulties., Material and Methods: In the present study we used highly sensitive and specific analytical methods for measuring LTs in the CSF as well as in urine samples from 20 patients with active MS and 20 control patients with noninflammatory neurological disorders., Results: LTB4 concentrations in CSF were almost twice as high in MS patients compared with controls (P < 0.001). CSF concentrations of the cysteinyl-LTs (LTC4, LTD4 and LTE4) as well as urinary LTE4 showed no significant differences compared with controls (P > 0.05). In addition, there was no significant association between CSF pleocytosis, clinical severity or time of disease onset., Conclusions: The increased concentration of LTB4 in the CSF of MS patients may indicate a biological importance for this mediator in MS.

The biochemical and biological characterization of a cytotoxic activity targeting macroglial cells (oligodendrocytes and astrocytes), in moncyte cultures and in CSF of a patient with multiple sclerosis, has previously been described. In further studies, cell-based tests have shown a good correlation between this glial cytotoxic (gliotoxic) activity, in CSF or in urine, and MS. We now present results obtained with urine samples from 102 MS patients, 51 patients with other neurological disease and 35 healthy subjects using a bioassay set up for the detection of an apoptosis-like effect induced in a glial cell-line. Significant gliotoxicity was detected in urine from 74/102 MS patients while only 4/51 neurological controls (P>0.001) and never in healthy subjects (P>0.001). Given the statistical tendency provided by this bioassay and its technical limitations for routine testing, it is now used for monitoring the molecular characterization of this 'gliotoxic factor'. Its replacement by a specific immunoassay could provide more accurate routine techniques for the detection of this biological marker in MS.

Implication of apoptosis in numerous physiological and pathological processes has resulted in the development of numerous methods to detect apoptosis, but none of them is adapted to all cell types. In this study, we induced apoptosis on murine immortalized astrocytes with urine from multiple sclerosis (MS) patients. Among techniques allowing the detection of apoptotic cells, only a few are adapted to adherent cells such as astrocytes. We compared several techniques (propidium iodide labelling and flow cytometry analysis, TUNEL and annexin V labelling in immunofluorescence, DNA ladder, ELISA tests to detect nucleosomes) in order to choose the method best adapted to our adherent cellular model and to discuss their practicability for the detection of apoptosis on adherent cells. For technical course, propidium iodide labelling followed by flow cytometry analysis as a quantitative technique, and TUNEL in IF (easier and quicker than propidium iodide) as a semiquantitative test were both retained as best adapted to our case. Moreover, in our model, we have observed that phosphatydilserine externalization and DNA fragmentation were concomittant after induction of apoptosis. Techniques studied in this article would allow an enlarged study of the apoptotic mechanism in several pathologies by culture of adherent cells sensitive to apoptosis in vitro.

Background: A significant correlation exists between disability and the volume of black holes (BHL VOL), defined as hypointense lesions on T1-weighted cranial magnetic resonance imaging. A consistent correlation has also been reported between urinary myelin basic protein-like material (MBPLM) and the transition toward secondary progression (SP) from relapsing-remitting (RR) multiple sclerosis (MS)., Objective: To improve the management of MS through a noninvasive and cost-effective test for monitoring disease activity or disease status., Design and Methods: From 662 patients with MS (86 with RR MS, 259 with SP MS without continued attacks, and 317 with SP MS with continued attacks), 24-hour urine samples were obtained at enrollment in the phase 3 Linomide (roquinimex) drug study. The urine specimens were analyzed for MBPLM and correlated with clinical features and findings on cranial magnetic resonance imaging., Results: Significant but weak correlations existed between urinary MBPLM and BHL VOL in all patients with MS (r = 0.114, P =.003; n = 662), patients with SP MS without attacks (r = 0.185, P =.003; n = 259), and all patients with SP MS (r = 0.122, P =.003; n = 576). No significant correlations were detected in the RR MS group or any of the disease groups or subgroups whose Expanded Disability Status Scale score was 5.0 or lower. In subgroup analysis, the most significant correlation was detected between urinary MBPLM after adjustment for creatinine and BHL VOL in patients with SP MS with an Expanded Disability Status Scale score of 5.5 or higher but without continued relapses (r = 0.417, P<.001; n = 138)., Conclusions: In patients with advanced SP MS, urinary MBPLM may possibly serve as an indicator of failed remission and axonal damage. Urinary MBPLM correlates with disease status in MS, especially the transition of RR MS to SP MS with advancing disability.

Multiple sclerosis (MS) is clinically heterogeneous and has an uncertain natural history. A high priority for more effective treatment of MS is an objective and feasible laboratory test for predicting the disease's course and response to treatments. Urinary myelin basic protein (MBP)-like material (MBPLM), so designated because it is immunoreactive as a cryptic epitope in peptide 83-89 of the human MBP molecule of 170 amino acids, is present in normal adults, remains normal in relapsing-remitting, but increases in progressive MS. In the present investigation, MBPLM was purified from urine and characterized. p-Cresol sulfate is the major component of urinary MBPLM. This conclusion is based on the following: (1) MBPLM and p-cresol sulfate both have a mass of 187 on negative scans by electrospray ionization mass spectrometry, the same fragments on tandem mass spectrometry of 80 (SO(-)(3)) and 107 (methylphenol), and similar profiles on multiple reaction monitoring; (2) (1)H and (13)C nuclear magnetic resonance spectroscopy revealed identical spectra for MBPLM and p-cresol sulfate; (3) purified p-cresol sulfate reacted in parallel with MBP peptide 83-89 in the same radioimmunoassay for MBPLM; and (4) p-cresol sulfate has the same behavior on preparative HPLC columns as urinary MBPLM. The unexpected immunochemical degeneracy permitting a cross-reaction between p-cresol sulfate and a peptide of an encephalitogenic myelin protein is postulated to be based on shared conformational features. The mechanisms by which urinary p-cresol sulfate, possibly derived from tyrosine-SO(4), reflects progressive worsening that is disabling in MS are unknown., (Copyright 2000 Academic Press.)

In multiple sclerosis (MS) gadolinium (Gd)-enhanced MRI activity correlates weakly with immunological markers of disease activity. We, therefore, tested the hypothesis that the poor correlation could be partly explained by the temporal profile of Gd enhancement. We measured urinary neopterin:creatinine ratios (neopt.:creat.(urine)) in 5 patients with active MS undergoing weekly Gd-enhanced MRI studies of the brain. The neopt.:creat.(urine) associated with new Gd-enhancing lesions (<8 days) was significantly higher than the ratio not associated with new Gd-enhancing lesions [mean(geometric) neopt.: creat.(urine) = 413 micromol/mol (range = 207-521) vs. 250 micromol/mol (range = 132-492), p = 0.03]. Pro-inflammatory immunological markers, which are probably produced early on in the life cycle of an active MS lesion, should preferably be correlated with newly enhancing lesions (<8 days). Failure to do this may explain the poor and unpredictable correlations between immunological markers and Gd-enhanced MRI activity, which cannot be accurately aged in cross-sectional and serial monthly MRI studies., (Copyright 2000 S. Karger AG, Basel)

Nitric oxide (NO) has been implicated in the immunopathogenesis of MS as a potential mediator of neuronal loss. To investigate the role of.NO in the development of progressive disease we measured the NO metabolites (nitrate and nitrite) and neopterin, in the urine of 129 patients with demyelinating disease (DD): 23 with clinically isolated syndromes compatible with demyelination and in 46 relapsing remitting (RR) and 60 patients with progressive MS. Eighty-nine of these 129 patients underwent Gd-enhanced MRI. In addition 58 normal control subjects (NC), 19 AIDS and 35 rheumatoid arthritis (RA) patients were studied. Patients with DD, AIDS and RA had significantly elevated urinary nitrate plus nitrite (nit : creat. urine) and neopterin (neopt : creat.urine) to creatinine ratios compared to NC subjects. (Median[25th - 75th%] nit : creat.urine: NC=1183[962 - 1365] vs DD=1245[875 - 2403], AIDS=1686[1231 - 2531], and RA=1950[1214 - 2726] mumol/mol, P<0.001 and median[25th - 75th%] neopt : creat.urine: NC=99[76 - 151] vs DD=163[119 - 266], AIDS=972[653 - 1456], and RA=389[257 - 623] mu mol/mol, P<0.001). Patients with early DD and RR MS had significantly elevated nit : creat.urine compared to patients with progressive MS (nit : creat. urine: 1612[1020 - 2733] vs 1159[790 - 1641] mu mol/mol, P=0.006). The nit : creat.urine and neopt : creat.urine did not correlate with clinical relapse or MRI activity. Excretion of.NO metabolites is increased in patients with early or relapsing-remitting disease.NO appears to be a double-edged sword, mediating tissue damage and modulating complex immunological functions which may be protective in MS.

A gradient eluent HPLC analysis in human plasma and urine was developed and validated for methylprednisolone (MP), its prodrug methylprednisolone-21-hemisuccinate (MPS) with the metabolites 6beta-hydroxy-6alpha-methylprednisolone (MPA), 20-hydroxymethylprednisolone (MPC), 6beta-hydroxy-20alpha-hydroxymethylprednisolone (MPB), 6beta-hydroxy-20beta-hydroxymethylprednisolone (MPE), 20-carboxymethylprednisolone (MPD), methylprednisolone-glucuronide (MPF) and 21-carboxymethylprednisolone (MPX). The column was Cp Spherisorb C8 5 microm, 250 mm x 4.6 mm I.D. (Chrompack, Bergen op Zoom, The Netherlands) with a guard column 75 mm x 2.1 mm, packed with pellicular reversed-phase. The eluent was a mixture of acetonitrile and 0.067 M KH2PO4 buffer, pH 4.5. At t=O, the eluent consisted of 2% acetonitrile and 98% buffer (v/v). Over the following 35 min the eluent changed linearly until it attained a composition of 50% acetonitrile and 50% buffer (v/v). At 37 min (t=37) the eluent was changed over 5 min to the initial composition, followed by equilibration over 3 min. The flow-rate was 1.5 ml/min and UV detection was achieved at 248 nm. Preliminary pharmacokinetic data were obtained from one patient who showed illustrative plasma concentration-time curves and renal excretion-time profiles after a short-lasting infusion (0.5 h) of 1 g of methylprednisolone hemisuccinate. The half-life of prodrug methylprednisolone-21-hemisuccinate (MPS) was 0.3 h, that of metabolite MPX (21-carboxy MP) was 0.4 h and that of the parent drug methylprednisolone (MP) was 1.4 h. The half-lives of the metabolites are almost similar (4 h). The main compounds in the urine are methylprednisolone hemisuccinate (prodrug, 15.0%), methylprednisolone (parent drug, 14.6%), metabolite MPD (20-carboxy, 11.7%), and metabolite MPB (13.2%). The renal clearance values of metabolites MPB, MPC and MPD are approximately 500 ml/min, that of MP is 100 ml/min.

Objectives: To determine levels of urinary myelin basic protein-like material (MBPLM) in patients with multiple sclerosis (MS) openly treated with interferon beta-1b and to correlate these with clinical changes., Background: Levels of urinary MBPLM correlate with the presence of the progressive phase of MS and with the disease burden detected on T2-weighted, cranial magnetic resonance imaging. Measurement of urinary MBPLM level may be a feasible test for monitoring or predicting response to therapeutic measures., Design and Methods: In a prospective study at one site, 166 patients with MS (131 with relapsing-remitting [RR] and 35 with secondary progressive [SP] disease) were treated for a minimum of 1 year and up to 3 years with interferon beta-1b and underwent assessment for neurologic disability (Expanded Disability Status Scale and Scripps Neurological Rating Scale) and change in disease subtype. Urine samples were obtained at 1219 of 1378 clinic visits, and urinary MBPLM level was determined and related to creatinine level to adjust for renal function., Results: Statistical analysis using the general linear models procedure confirmed previous findings that the level of urinary MBPLM related to urinary creatinine level (MBPLM/creatinine) was higher (P<.001) in patients with SP than RR MS. Of the 131 patients with RR MS, SP disease developed in 13 during the observation period. Compared with those in the RR group, the RR to SP group had a higher level (P<.001) of urinary MBPLM and did not differ from the SP group., Conclusions: The level of urinary MBPLM is higher in SP MS than RR MS but not in RR MS that converts to SP MS. Level of urinary MBPLM may permit the examination of treatment tested to prevent RR disease from becoming progressive.

In the present study metabolites of methylprednisolone were detected using gradient elution high-performance liquid chromatography. Separation was performed by a Cp Spherisorb ODS 5 microm (250 mmx4.6 mm I.D.) column, connected to a guard column, packed with pellicular reversed phase. The mobile phase was a mixture of acetonitrile and 1% acetic acid in water. At t = 0, this phase consisted of 2% acetonitrile and 98% acetic acid 1% in water (v/v). During the following 35 min the phase changed linearly until it attained a composition of acetonitrile-buffer (50:50, v/v). At 40 min (t = 40) the mobile phase was changed over 5 min to the initial composition, followed by equilibration during 2 min. The flow-rate was 1.5 ml/min. UV detection was achieved at 248 nm. We have isolated the respective compounds with the most abundant concentration and suggested their chemical structure based on NMR, IR, UV, MS, retention behaviour and melting points. The c/, stereochemistry could not be solved in this study. The overall picture of the metabolic pathways of methylprednisolone is apparently simple: reduction of the C20 carbonyl group and further oxidation of the C20-C21 side chain (into C21-COOH and C20-COOH), in competition with or additional to the oxidation at the C6-position.

The evaluation of free light chains kappa in urine was performed in 77 cases of multiple sclerosis, including 52 patients before and after treatment with 2 CDA and in 25 patients before and after therapy with high doses of prednisone. The high variations in the level of free kappa chains indicate a limited diagnostic value, and only for cases with very high level. We have found effect of 2 CDA therapy in chronic progressive MS group on free kappa light chain value. A significant effect of prednisone treatment was observed in early onset cases of multiple sclerosis and in cases with clinical improvement after therapy. In conclusion, the study suggests that urinary free light chains level may be considered as one of markers for monitoring of the effect of therapy on the activity of the immunological processes in multiple sclerosis.

We measured free kappa (kappa) and lambda (lambda) light chains in urine from patients with definite multiple sclerosis (MS), other neurologic diseases (OND), and normal controls by using an enzyme linked immunosorbent assay. Both kappa and lambda light chains were higher in MS than OND or controls. In seven of eight relapsing-remitting (R-R) MS patients serial studies showed that urinary kappa chains were elevated during periods of worsening, and decreased during clinical recovery. In contrast, the levels of kappa chains did not correlate with clinical activity in 10 progressive (P) MS patients. Further correlation of urinary light chains with neurologic evaluations in R-R and P MS patients over a longer period are needed to determine their clinical and biological relevance.

Numerous markers of disease activity, representing different aspects of the inflammatory cascade and pathogenic process in multiple sclerosis, can be detected in the urine. Urinary monitoring provides distinct advantages over blood and cerebrospinal fluid: it is easier to collect, allows frequent sampling and acts as a natural integrator by capturing the excretion of a substance over a prolonged period of time. We will discuss the principles, advantages, and pitfalls of urinary monitoring in relationship to multiple sclerosis.

Urinary myelin basic protein-like material (MBPLM) represents material which is cross-reactive with a cryptic epitope in peptide 84-89 of human myelin basic protein. While normally present at moderate levels in the adult, these levels rise higher in patients who have secondary progressive multiple sclerosis (MS). The increase in urine MBPLM correlates with the burden of disease detected by T2-weighted cranial magnetic resonance imaging. There is no correlation between urinary MBPLM and acute disease activity in relapsing-remitting MS. The first major need for improving the clinical utility of measurements of MBPLM in urine in MS patients is to delineate its exact chemical features so that assays may be improved and a potential biological role of the MBPLM better understood. The second major task is to apply the group data accumulated and apply them to individual patients. This could prove to be means to individually direct treatment and determine its effectiveness.

The regulatory region of progressive multifocal leukoencephalopathy-type JC virus (JCV) is rearranged in each host by a process of deletion and duplication. Of the more than 40 that have been examined, no two regulatory regions have been rearranged identically in the brain. The substrate for this rearrangement appears to be a highly stable archetypal regulatory region excreted in the urine. Its role as the transmissible form of the virus, although inferred, has never been proven. We have now amplified by PCR and cycle-sequenced the regulatory regions from 48 urinary strains of the virus. We find that the urinary form of the regulatory region is not entirely stable. Short deletions and duplications in the range of 2-16 bp were observed in seven of these strains. One of these, an inverted repeat, is a pattern of rearrangement not yet found in the brain. Two others (#208 and 230) showed a 2-bp deletion at position nos. 221 and 222, and an unusual mutation at position no. 219. These two urines were collected in different states of the USA at different times and analysed months apart. It is very unlikely that these unusual changes represent sample contamination or that they arose independently. This finding indicates that archetypal forms of the JCV regulatory region are infectious, despite their relative inactivity in tissue culture. While changes in the archetypal structure can be found, it is clear that rearrangements in the kidney are rare or rarely infectious.