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Objectives: To develop a model of disease progression using multiple sclerosis (MS) as an exemplar., Study Design and Settings: Two observational cohorts, the University of Wales MS (UoWMS), UK (1976), and British Columbia MS (BCMS) database, Canada (1980), with longitudinal disability data [the Expanded Disability Status Scale (EDSS)] were used; individuals potentially eligible for MS disease-modifying drugs treatments, but who were unexposed, were selected. Multilevel modeling was used to estimate the EDSS trajectory over time in one data set and validated in the other; challenges addressed included the choice and function of time axis, complex observation-level variation, adjustments for MS relapses, and autocorrelation., Results: The best-fitting model for the UoWMS cohort (404 individuals, and 2,290 EDSS observations) included a nonlinear function of time since onset. Measurement error decreased over time and ad hoc methods reduced autocorrelation and the effect of relapse. Replication within the BCMS cohort (978 individuals and 7,335 EDSS observations) led to a model with similar time (years) coefficients, time [0.22 (95% confidence interval {CI}: 0.19, 0.26), 0.16 (95% CI: 0.10, 0.22)] and log time [-0.13 (95% CI: -0.39, 0.14), -0.15 (95% CI: -0.70, 0.40)] for BCMS and UoWMS, respectively., Conclusion: It is possible to develop robust models of disability progression for chronic disease. However, explicit validation is important given the complex methodological challenges faced., (Crown Copyright © 2015. Published by Elsevier Inc. All rights reserved.)

This review describes the state of the art for the use of laboratory testing in myasthenia gravis. The review brings a detailed description of the different clinical forms of auto-immune myasthenia and of the Lambert Eaton Myasthenic Syndrome (LEMS). They stress the differences between the different forms of acquired (auto-immune) myasthenia. Then they present a summary of the different antibodies found in the disease. They insist on the advantage of the RIPA assay to measure antibodies to the acetylcholine receptor. They stress the different types of contribution of each of these antibodies to the clinical diagnosis. They also describe the methods to measure each of the specific antibodies that have recently permitted to split the diagnosis: Abs to omega-conotoxin receptor in Lambert Eaton Myasthenic Syndrome (LEMS), abs to the acetylcholine receptor (AchR) in MG, Abs to muscle specific tyrosine kinase (MuSK) in Ab negative MG, and Abs to low molecular weight receptor related low-density lipo protein-4 (LRP-4). They also broach over the striated antibodies, less frequent and clinically less useful such as anti-titin, -ryanodine, -agrin and -rapsyn. This represent a 360° view of the field as presented in Toronto in October 2014., (Copyright © 2015. Published by Elsevier B.V.)

This review describes the state of the art for the use of laboratory testing in myasthenia gravis. The review brings a detailed description of the different clinical forms of auto-immune myasthenia and of the Lambert Eaton Myasthenic Syndrome (LEMS). The stress the differences between the different forms of acquired (auto-immune) myasthenia. Then they present a summary of the different antibodies found in the disease. They insist on the advantage of the RIPA assay to measure antibodies to the acetylcholine receptor. They stress the different types of contribution of each of these antibodies to the clinical diagnosis. They also describe the methods to measure each of the specific antibodies that have recently permitted to split the diagnosis: Abs to omega-conotoxin receptor in Lambert Eaton Myasthenic Syndrome (LEMS), abs to the acetylcholine receptor (AchR) in MG, Abs to muscle specific tyrosine kinase (MuSK) in Ab negative MG, and Abs to low molecular weight receptor related low-density lipo protein-4 (LRP-4). They also broach over the striated antibodies, less frequent and clinically less useful such as anti-titin, -ryanodine, -agrin and -rapsyn. This represent a 360° view of the field as presented in Toronto in October 2014., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Multiple sclerosis (MS) is associated with chronic degeneration of the central nervous system and may cause permanent neurological problems and considerable disability. While its causes remain unclear, its extensive phenotypic variability makes its prognosis and treatment difficult. The identification of serum proteomic biomarkers of MS progression could further our understanding of the molecular mechanisms related to MS disease processes. In the current study, we used isobaric tagging for relative and absolute protein quantification (iTRAQ) methodology and advanced multivariate statistical analysis to quantify and identify potential serum biomarker proteins of MS progression. We identified a panel of 11 proteins and combined them into a classifier that best classified samples into the two disease groups. The estimated area under the receiver operating curve of this classifier was 0.88 (p-value=0.017), with 86% sensitivity and specificity. The identified proteins encompassed processes related to inflammation, opsonization, and complement activation. Results from this study are in particular valuable to design a targeted Multiple Reaction Monitoring mass spectrometry based (MRM-MS) assay to conduct an external validation in an independent and larger cohort of patients. Validated biomarkers may result in the development of a minimally-invasive tool to monitor MS progression and complement current clinical practices., Biological Significance: A hallmark of multiple sclerosis is the unpredictable disease course (progression). There are currently no clinically useful biomarkers of MS disease progression; most work has focused on the analysis of CSF, which requires an invasive procedure. Here, we explore the potential of proteomics to identify panels of serum biomarkers of disease progression in MS. By comparing the protein signatures of two challenging to obtain, but well-defined, MS phenotypic groups at the extremes of progression (benign and aggressive cases of MS), we identified proteins that encompass processes related to inflammation, opsonization, and complement activation. Findings require validation, but are an important step on the pathway to clinically useful biomarker discovery. This article is part of a Special Issue entitled: Protein dynamics in health and disease. Guest Editors: Pierre Thibault and Anne-Claude Gingras., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The appearance of neutralizing antibodies (NAbs) has significant clinical and regulatory consequences for interferons in patients with multiple sclerosis (MS). In a double blind, randomized clinical trial, 84 patients with relapsing remitting MS were enrolled in a 24month study period. Patients were randomly assigned into two groups receiving 30mcg weekly intramuscular injections of either Avonex® (Biogen Idec, USA; 42 patients) or CinnoVex® (CinnaGen Co, Iran; 42 patients). NAb titer was drawn for all patients every 6months and assayed using cytopathic effect assay (CPE) method in Tehran, Iran. To validate the measure done in the Iranian lab, 45 sera with adequate volume and proper storing condition were selected and sent to be rechecked using luciferase reporter gene assay (LA) method for verification in 2 phases in Vancouver, Canada. The cut-off point of 20 TRU was considered for positivity. The two labs found the same three samples to be positive (2 samples from patients received Avonex and 1 received CinnoVex) and 42 to be negative. They had the following values using the Kawade formula as recommended by international standards; 2238, 89 and 302 (TRu/ml) using CPE assay versus 2464, 290 and 169 (TRu/ml) using LA method. As similar results were obtained from CinnoVex or Avonex in our study, we suggest that both medications will have a similar immunogenetic profile., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The objective of this study was to assess the short-term efficacy and safety of terbutaline, a beta2-adrenergic agonist, in patients with myasthenia gravis (MG) in a randomized, double-blind, placebo-controlled, crossover study. The primary endpoint for efficacy was a reduction of at least 3 points in the quantitative MG score (QMGS). Secondary endpoints included changes in the functional disability scale (FDS), forced vital capacity (FVC), grip strength, anti-acetylcholine receptor (AChR) antibody levels and decremental response. During the terbutaline phase, five of eight (63%) patients had an improvement in the QMGS of 3.0 or greater, while 3/8 (38%) patients had improvement in the FDS of one grade. No improvement was seen during the placebo period. Statistical analysis using Wilcoxon signed-rank test confirmed that terbutaline treatment resulted in a significant improvement in QMGS. There was no change in FVC, grip strength or anti-AChR antibody levels, but there was an improvement in the decremental response during terbutaline phase. Terbutaline was well-tolerated in all study subjects. We conclude that terbutaline may be an effective adjunct therapy in a subset of patients with myasthenia, although confirmation with larger trials will be required.

Multiple sclerosis (MS) is characterised by focal areas that undergo cycles of demyelination and remyelination. Although conventional magnetic resonance imaging is very effective in localising areas of damage, these techniques are not pathology specific. A newer technique, T(2) relaxation, can separate water from brain into three compartments: (1) a long T(2) component (>2 s) arising from CSF, (2) an intermediate T(2) component (~80 ms) attributed to intra- and extra-cellular water and (3) a short T(2) component (~20 ms) assigned to water trapped in between the myelin bilayers (termed myelin water). Histological evidence shows that myelin water is a specific marker of myelination. The goal of this work was to follow changes in total water content (WC) and myelin water fraction (MWF) in evolving MS lesions over one year. Multi-echo T(2) relaxation data was collected and used to measure water content and myelin water fraction from three new MS lesions in two patients. WC increased in the three large (>1 cm(3)) lesions at lesion appearance and remained elevated in the central core. Two lesions showed low MWF in the core suggesting demyelination upon first appearance. At later time points, one lesion showed a decrease in volume of low MWF, reflecting remyelination whereas the volume of low MWF in the other lesion core remained constant. This work provides evidence that MWF and WC can monitor demyelination and remyelination in MS.

Administered proteins are inherently immunogenic, which may influence their efficacy or safety when used therapeutically. A review of the published literature was performed to compare and evaluate the development and consequences of antibodies against therapeutic protein agents for the treatment of multiple sclerosis (MS). Interferon beta (IFNbeta), glatiramer acetate (GA), and natalizumab are all protein-based therapeutic agents approved to treat MS and are associated with the development of antibodies. Both binding antibodies and neutralizing antibodies (NAbs) develop to varying degrees in patients treated with any of the formulations of IFNbeta. Comparison between studies is complicated by differences in methods, assays, criteria for determining NAb positivity, treatment duration, and fluctuation of NAb status. Despite these confounding factors, current data indicate that high-titer persistent NAbs may be relevant in terms of their effect on IFNbeta bioavailability and bioefficacy. GA-reactive antibodies developed in a high proportion of GA-treated patients, but the clinical relevance of these antibodies remains to be established. Immunogenicity against natalizumab was associated with reduced efficacy and increased incidence of infusion reactions. Other emerging monoclonal antibody therapeutics have also been associated with the development of antibodies. Experience with generic biosimilars of other protein therapeutics suggests that the immunogenicity of generic biosimilar agents cannot be assumed and must be established for each formulation.

Neutralizing antibodies (NAbs) can occur in some multiple sclerosis (MS) patients receiving interferon beta (IFNbeta) therapy. NAbs reduce drug bioavailabity and high NAb titers reduce drug efficacy. We describe the validation of the R. Farrell and G. Giovannoni luciferase reporter gene assay to measure NAbs to INFbeta. We assayed 163 sera from IFNbeta treated MS patients with an optimized luciferase method and compared the results to those obtained with the reference cytopathic effect (CPE) method using A549 cells and an encephalomyocarditis virus (EMCV). Binding antibodies (BAbs) were measured using a capture ELISA as a screening test for NAbs in the CPE assay. NAb status measured by the luciferase and the ELISA/CPE method did not yield a significant difference. Log10 NAb titers obtained from the luciferase assay and the A549/EMCV CPE methods correlated very well. The inter-assay coefficient of variation for titers was between 17.8-29.3%, and the intra-assay coefficient of variation was between 6.3-15.2%. The luciferase assay is reliable, appropriately sensitive and requires less time than the currently available NAb methods.

The HTLV-1 virus produces a progressive inflammatory, and then degenerative, myelopathy which evolves progressively from onset. HTLV-1 in endemic in populations which are recognized as having low risk of multiple sclerosis . Multiple sclerosis generally evolves as a relapsing-remitting disease and affects predominantly Caucasians. In Caucasians, HAM/TSP can be marked by fluctuations as well as relapses. In Asians MS affects preferentially the spinal cord. The author hypothesizes that population selection through environmental factors has pushed the immune response of Caucasians towards generating relapsing-remitting disease and that of Primordial populations towards progressive disease. HTLV-1 endemicity being the marker of Primordial populations and its absence that of Caucasians.

The author participated very early in the use of immunosuppressors in the treatment of multiple sclerosis. He reviews evidence which support their use. IV Methylprednisolone, azathioprine and mitoxantrone are supported in their use by evidence of a level appropriate to the date of their generation while Cyclosporine A and Cyclophosphamide are not. The author also reviews the benefits and side effects of each of these medications, insisting on a practical approach to their use. The author concludes that since immunomodulators have been approved, the use of the immunosuppressors has been reduced, however there is a strong possibility that their use will be rekindled in association with immunomodulatory medications.

Background: Recent guidelines have recommended the use of validated assays for the measurement of neutralizing antibodies (NABs) to interferon beta (IFNbeta) in patients with multiple sclerosis (MS). In an attempt of validation, we studied the analytical performance of a bioassay based on antiviral cytopathic effect (CPE) using WISH cells and the vesicular stomatitis virus (WISH/VSV CPE)., Methods: NAB titres measured with the WISH/VSV CPE assay in 63 sera from IFNbeta-treated MS patients were compared to those obtained with the reference CPE method using A549 cells and the encephalomyocarditis virus. Binding antibodies (BABs) were measured using a capture ELISA as a screening test for NABs., Results: No false-negative BAB was obtained in our patients. The between-run coefficients of variation (CVs) determined with log10 titres of the NIH anti-IFNbeta (G038-501-572) yielded good results (

The efficacy of interferon (IFN) beta has been shown in several placebo-controlled, parallel-group studies in relapsing-remitting multiple sclerosis (RRMS). PRISMS, the largest such study to date, clearly demonstrated the efficacy of IFN beta-1a on all outcome measures over 2 years during the placebo-controlled, parallel-group phase. However, this study's placebo-crossover design also provided us with a unique opportunity to conduct a prospective within-group assessment, eliminating the impact of inter-patient variability. At the start of year 3, patients receiving placebo during years 1-2 were re-randomized in a dose-blinded fashion to receive IFN beta-1a, 22 or 44 mcg subcutaneously three times weekly, during years 3 and 4. Clinic visits occurred 3-6 monthly and T2 MRI scans were obtained after 1 and 2 years on therapy. Comparison of the mean relapse count per patient over 2 years (the primary outcome measure) during time on placebo (years 1 and 2) with that during active treatment (years 3 and 4) revealed a decrease from 2.6 to 1.2 in both dose groups (54% relative reduction; p<0.001). Disability progression, T2 MRI lesion activity and accumulation of T2 lesion burden were also significantly improved with therapy (p<0.01). No new safety issues were noted. These data provide further support for IFN beta-1a's efficacy in RRMS. The ability to detect significant treatment effects with reduced patient numbers in this type of before/after analysis, may be due to the reduction in inter-patient variability.

In myasthenia gravis (MG), presence of pathogenic acetylcholine receptor antibodies (AchRAb) and thymic pathology are related. Our study assesses the long-term clinical outcome of thymectomy as a function of thymic pathologies, AchRAb levels in blood and the ability of immunocytes to secrete AchRAb ex vivo. We found that those patients who have thymocytes that secrete AchRAb in vitro have a greater decrease in their need for Mestinon over the 60 months of follow-up than the non-secretors (p = 0.04). Those patients whose cultured peripheral blood mononuclear cells (PBMC) secreted AchRAb also had a greater decrease in Mestinon requirements over the 60 months of follow-up than the non-secretors (p = 0.05), however, differences between the two groups at each individual assessment were not statistically significant. Serial post-op AchRAb measurements in sera of thymocyte and PBMC non-secretors showed no change from pre-op levels (< 10 nmol/l), while secretors had a reduction of more than 80% from pre-op levels, starting 6 months post-op. We also found that results of PBMC culture at time of thymectomy is a better predictor of response to thymectomy than serum AchRAb levels but inferior to results of thymocytes cultures. Patients with thymoma had the highest proportion of individuals secreting AchRAb from thymocytes in culture (78%), the shortest duration of disease (9 months) and showed the greatest trend toward improvement in clinical signs and symptoms post-op. We have thus demonstrated the long held suspicion that removal of actively secreting cells from the thymus at an early time in the disease confers the greatest benefit., (Copyright 2002 Elsevier Science B.V.)

We have investigated lymphocyte subpopulations and blood mononuclear cell (MNC) adhesion to activated endothelial monolayers in patients with human T lymphotropic virus type I (HTLV-I) associated myelopathy (HAM), in HTLV-I asymptomatic carriers (carriers), and in seronegative controls. HAM patients and carriers had higher levels of CD4(+)CD29(+) "memory cells" than controls (P < 0.05). The percentage of CD3(+)CD27(-) "primed T cell" was elevated in patients with HAM (P < 0.05), but not in carriers. HAM patients had higher levels of CD8(+)CD57(+) "cytotoxic cells" (P < 0.05) than controls and carriers. The percentages of CD4(+) cells coexpressing activation markers HLA-DR and CD25, and of CD8(+) cells expressing HLA-DR, were significantly higher in HAM patients and carriers than in controls. Functional experiments indicated that MNC from HAM patients adhered more to activated endothelial monolayers than MNC from carriers or controls. Blocking studies demonstrated that the adhesion molecules VLA-4 and ICAM-1 and also L-selectin all contributed to increased binding. Analysis of expression of molecules involved in adhesion indicated that in HAM patients, L-selectin (CD62L) expression on CD4(+) and CD8(+) subsets was lower than in controls. Interestingly, HAM patients had a lower percentage of CD4(+) subsets expressing L-selectin than carriers (P < 0.05). In contrast, the percentage of CD4(+) and CD8(+) cells expressing VLA-4 (CD49d) was found to be higher in both HAM patients and carriers compared with controls. After 2 days in culture without mitogen, the percentage of T cells expressing ICAM-1 (CD54) increased in culture in carriers and more profoundly in HAM, but not in controls (P < 0. 05). After culture, T cells expressing the early activation antigen CD69 were also increased in HAM and carriers (P < 0.05) but not in controls. Interestingly, the levels of CD8(+) cells coexpressing activation antigen HLA-DR and CD38 were higher in HAM patients compared with both carriers and controls (P < 0.05) after culture. These findings are consistent with the observations that HTLV-I produces chronic lymphocyte activation with increased adhesion. This may be sufficient to initiate events leading to central nervous system inflammation and ultimately to HAM., (Copyright 1999 Academic Press.)

We describe a simple solid-phase radioimmunoassay (RIA) to detect IgG based on competitive binding between radiolabeled and unlabeled IgG for anti-IgG antibody physically adsorbed to the wells of polyvinyl microtiter plates. The assay is sensitive (1 ng), rapid, and is particularly suited for studies of in vitro IgG secretion by human peripheral blood lymphocytes, since such studies require large numbers of cultures. Conditions which permit measurement, by means of this assay, of helper and suppressor T cell effects on IgG production by human B cells in culture are described.

261 multiple sclerosis (MS) patients were HLA-A and -B typed and 94 were HLA-D typed. The results were compared to those of controls typed for HLA-A; HLA-B (356) and HLA-D (113). We confirm and extend earlier findings (Oger et al. 1980b) that some phenotypes could modulate the expression of the MS susceptibility gene linked to B7-DR2: DR3 was found together with DR2 in 12/94 MS and only 3/113 controls and could be marker for an "augmentor" gene. In contrast, B35 and DR1 as well as B12 and DR7 could be markers of protector genes. We compared typing results of patients subgrouped on clinical features. 61 patients with progressive evolution showed increased A1, A1-B8, B8-DR3 and A1-B8-DR3 when compared to 200 patients with remitting evolution. When compared to controls both groups showed increased B7 but only the remitting group showed increased DR2. 71 patients with "benign MS" showed increased B7-DR2 and A3-B7-DR2. 54 patients with "severe disease" showed increased DR3 and A1-B8-DR3 when compared to controls. Both groups showed increased B7 (49.2% and 44.4% versus 25.5% for controls). 120 patients treated greater than 5 years with azathioprine were divided into "no progression" and "progression" while treated. Both groups showed increased B7 when compared to controls. DR2 was increased only in the "no progression" group. B8-DR3 and A1-B8-DR3 were found increased in the "progression" group only. We conclude that two forms of MS exist with different HLA profiles.

In vitro modulation of human T lymphocyte surface differentiation antigens T3, T8, and T4, by their respective monoclonal antibodies, was studied as a function of donor age. Kinetic studies performed on lymphocytes from young adults indicated that modulation is dependent on concentration of antibody used and duration of culture. OKT3 modulates T3 rapidly (maximum at less than 24 hr) and relatively completely (79% at the highest concentration of antibody used). By 48 hr, regeneration of the T3 antigen is apparent. T8 modulation by OKT8 is slower (continued modulation at 48 hr) and less complete than is T3 modulation by OKT3. OKT4 does not modulate the T4 antigen. In elderly individuals modulation of T3 by OKT3 is preserved whereas modulation of T8 by OKT8 is significantly reduced (24 +/- 8% at 48 hr vs 53 +/- 4% for young controls). These observations document further age-related changes in properties of human T suppressor cells.

PWM-induced differentiation of human PBMC into immunoglobulin (Ig) secreting cells is a widely used model of in vivo IgG secretion. We examined the relationship between the amount of IgG secreted in PBMC cultures obtained from individuals who consistently secrete either very high (HR) or very low amounts (LR) of IgG and various in vitro immune function assays. The PBMC obtained from LR contained a higher ratio of cells expressing the T-suppressor/inducer to T-helper/inducer phenotype. The autologous mixed lymphocyte response was lower and the amount of in vivo radiation sensitive suppression was higher in the LR than in the HR. LR E- cells secreted less IgG than the HR E- cells when both were mixed with heterologous HR E+ cells. Monocyte depletion reduced IgG secretion in both LR and HR. These results suggest that each of the subsets Ts, Th (Thi, Tsi), and B lymphocytes are involved in the regulation of PWM-induced Ig secretion and that the functions of each of these subsets differ in HR and LR individuals.