The Effect of Disease Modifying Therapies on Disease Progression in Patients with Relapsing-Remitting Multiple Sclerosis: A Systematic Review and Meta-Analysis.

A number of officially approved disease-modifying drugs (DMD) are currently available for the early intervention in patients with relapsing-remitting multiple sclerosis (RRMS). The aim of the present study was to systematically evaluate the effect of DMDs on disability progression in RRMS.We performed a systematic review on MEDLINE and SCOPUS databases to include all available placebo-controlled randomized clinical trials (RCTs) of RRMS patients that reported absolute numbers or percentages of disability progression during each study period. Observational studies, case series, case reports, RCTs without placebo subgroups and studies reporting the use of RRMS therapies that are not still officially approved were excluded. Risk ratios (RRs) were calculated in each study protocol to express the comparison of disability progression in RRMS patients treated with a DMD and those RRMS patients receiving placebo. The mixed-effects model was used to calculate both the pooled point estimate in each subgroup and the overall estimates.DMDs for RRMS were found to have a significantly lower risk of disability progression compared to placebo (RR = 0.72, 95%CI: 0.66-0.79; p<0.001), with no evidence of heterogeneity or publication bias. In subsequent subgroup analyses, neither dichotomization of DMDs as "first" and "second" line RRMS therapies [(RR = 0.72, 95% CI = 0.65-0.80) vs. (RR = 0.72, 95% = 0.57-0.91); p = 0.96] nor the route of administration (injectable or oral) [RR = 0.75 (95% CI = 0.64-0.87) vs. RR = 0.74 (95% CI = 0.66-0.83); p = 0.92] had a differential effect on the risk of disability progression. Either considerable (5-20%) or significant (>20%) rates of loss to follow-up were reported in many study protocols, while financial and/or other support from pharmaceutical industries with a clear conflict of interest on the study outcomes was documented in all included studies.Available DMD are effective in reducing disability progression in patients with RRMS, independently of the route of administration and their classification as "first" or "second" line therapies. Attrition bias needs to be taken into account in the interpretation of these findings

The effect of disease-modifying therapies on brain atrophy in patients with clinically isolated syndrome: a systematic review and meta-analysis

Objectives: Brain atrophy is associated with cognitive deficits in patients with clinically isolated syndrome (CIS) and can predict conversion to clinical definite multiple sclerosis. The aim of the present meta-analysis was to evaluate the effect of disease-modifying drugs (DMDs) on brain atrophy in patients with CIS. Methods: Eligible placebo-control randomized clinical trials of patients with CIS that had reported changes in brain volume during the study period were identified by searching the MEDLINE, SCOPUS, and Cochrane Central Register of Controlled Trials (CENTRAL) databases. This meta-analysis adopted the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines for systematic reviews and meta-analyses. Results: Three eligible studies were identified, comprising 1362 patients. The mean percentage change in brain volume was found to be significantly lower in DMD-treated patients versus placebo-treated subgroups (standardized mean difference [SMD]: = −0.13, 95% confidence interval [CI]: −0.25, 0.01; p = 0.04). In the subgroup analysis of the two studies that provided data on brain-volume changes for the first (0–12 months) and second (13–24 months) year of treatment, DMD attenuated brain-volume loss in comparison with placebo during the second year (SMD = −0.25; 95% CI: −0.43, −0.07; p < 0.001), but not during the first year of treatment (SMD = −0.01; 95% CI: −0.27, 0.24; p = 0.93). No evidence of heterogeneity was found between estimates, while funnel-plot inspection revealed no evidence of publication bias. Conclusions: DMDs appear to attenuate brain atrophy over time in patients with CIS. The effect of DMDs on brain-volume loss is evident after the first year of treatment

Anti-AQP1 antibody binding to synthetic peptides corresponding to AQP1 extracellular and cytoplasmic domains.

<p><b>A.</b> Synthetic peptides corresponding to human AQP1 extracellular loops A (residues 36–49), C (116–137), and E (183–188, 199–210) or to the intracellular N-terminus (1–22), loop B (81–100), and the C-terminus (248–270) are shown (from Uniprot/Swissprot entry P29972, for the human AQP1). Extracellular residues are shown in bold and underlined. Loop-E is formed by two segments separated by a 14 residue gap; in “pept-Loop-E”, 10 of these 14 residues (between TG and SE) were omitted (marked by:..). In the first 4 peptides, an extra tyrosine residue was added to the N-terminus for future radioiodination studies. Below the AQP1 peptide sequences are shown the corresponding AQP4 sequences; (*), identical amino acid residues; (:) and (.), homologous residues. <b>B.</b> Peptide mapping with anti-AQP1 positive sera. 22 sera from patients with known clinical characteristics (17 NMOsd and 5 MS) were tested for binding to 6 synthetic peptides corresponding to the 3 extracellular loops (loops A, C and E) and to the 3 cytoplasmic segments (N-terminal, Loop-B and C-terminal) by an ELISA assay. Positive values are considered those above O.D.₄₅₀ 0.45 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074773#s2" target="_blank">Methods</a>). It is shown that most NMOsd sera bind to the Loop-A peptide.</p

Detection of binding of anti-AQP1 antibody to denatured AQP1 by Western blotting.

<p>Yeast-expressed AQP1 was electrophoresed and transferred onto nitrocellulose membranes, which were then incubated with the test sera (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074773#s2" target="_blank">Methods</a>). Lanes 1-3: Three sera from healthy controls at dilutions 1/250, 1/60, and 1/30, respectively; lane 4: commercial rabbit anti-AQP1 antibody; lanes 5-8: representative anti-AQP1-positive sera with titers of 132, 25, 10.5, and 5.8 nM, at dilutions of 1/500, 1/250, 1/30, and 1/30, respectively. None of the test anti-AQP1 sera bound to the control protein MuSK (not shown).</p

Laboratory and clinical data for anti-AQP1 seropositive patients.

<p>F, female; LETM, longitudinally extensive transverse myelitis; M, male; MS, multiple sclerosis; NMO, neuromyelitis optica; NT, not tested.</p>a<p>All 22 patients tested negative for serum anti-AQP-4 autoantibodies in the two-step RIPA and confirmed by both a commercial and an in-house CBA.</p>b<p>The anti-AQP1 positivity of the sera was also confirmed by the more sensitive two-step RIPA for anti-AQP1 antibodies (not shown).</p>c<p>According to the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074773#s2" target="_blank">Methods</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074773#pone-0074773-g008" target="_blank">Figure 8</a>.</p>d<p>Calculated by multiplying anti-AQP1 titers (in 6^th column) by the percentages of the extracellularly binding antibodies (in 7^th column).</p>e<p>Binding of sera to synthetic peptides corresponding to AQP1 segments (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074773#pone-0074773-g009" target="_blank">Figure 9B</a>). Underlined loops are extracellular.</p

Anti-AQP1 specificity of the identified autoantibodies.

<p>(A) Patient’s autoantibodies recognize the AQP1 moiety of AQP1-GST. Five sera that had tested positive for binding to the AQP1-GST fusion protein were preincubated with an excess of GST immobilized on Sepharose-glutathione beads, then were tested by RIPA using ¹²⁵I-streptavidin labeled AQP1-GST. (B) Binding of anti-AQP1 autoantibodies is specifically inhibited by an extract from AQP1-expressing HEK293 cells, but not control HEK293 cells. Four anti-AQP1-positive sera were preincubated with extracts prepared from either EGFP-transfected or AQP1-GFP-transfected HEK293 cells, then were tested by RIPA for binding to the commercial AQP1 preparation. (C) Binding of anti-AQP1 autoantibodies is specifically inhibited by yeast-expressed human AQP1. Four exclusively anti-AQP1-positive sera were preincubated with human AQP1 or AQP4 that had been expressed in yeast and purified or with BSA as control, then were tested by RIPA using ¹²⁵I-streptavidin-labeled commercial AQP1-GST fusion protein. (D) AQP1 autoantibody binding is independent of the source of AQP1. Both the commercial AQP1-GST fusion protein and the in house AQP1 purified from yeast were biotinylated, indirectly labeled by preincubation with ¹²⁵I-streptavidin, and used in the RIPA. Five anti-AQP1-positive sera and one serum sample from a healthy control (HC) were tested.</p

Liver powder removes anti-AQP1 antibodies.

<p>Two exclusively anti-AQP1-positive and two exclusively anti-AQP4-positive serum samples were pretreated with guinea pig liver powder, then the supernatants were tested by RIPA using indirectly radiolabeled AQP1 (left panel) or AQP4 (right panel). Key: +, pretreated serum; -, untreated serum.</p

Detection of anti-AQP1 antibodies by an ELISA with immobilized purified human yeast-expressed AQP1.

<p>Sera previously tested by RIPA for AQP1 antibodies were tested for binding to immobilized AQP1 by ELISA. First column contains 31 sera found positive by the RIPA (including a double-positive anti-AQP1/AQP4, empty square, and 3 sera from anti-AQP1-positive MS patients, empty circles). The following 3 columns contain 5 anti-AQP4-positive/anti-AQP1-negative sera, 30 sera from anti-AQP1-negative MS patients and 44 sera from healthy controls). The dashed horizontal line denotes the cut-off (O.D.450: 0.36) for positive values.</p

Anti-AQP1 antibodies and anti-AQP4 antibodies do not bind to the other antigen.

<p>(A and B). Search for cross-reactivity of anti-AQP1 and anti-AQP4 antibodies with AQP4 and AQP1, respectively. Three (A) or five (B) double-positive serum samples were incubated with immobilized AQP1 (A) or AQP4 (B) to immunoadsorb the corresponding antibodies, then were tested by RIPA for binding to ¹²⁵I-labeled AQP1 or AQP4 (white bars) in parallel with the untreated sera (black bars). HC, healthy control. (C). Lack of correlation of the amount of radiolabeled AQP1 precipitated by anti-AQP1 antibodies (y axis) or AQP4 (x axis) precipitated anti-AQP4 antibodies by double-positive sera in identical regular RIPAs performed using the two labeled antigens.</p

Determination of the percentage of antibodies directed against the extracellular domain of membrane-embedded AQP1.

<p>Four anti-AQP1-positive sera were left untreated or were preincubated with increasing numbers of AQP1-GFP- (filled symbols) or EGFP-transfected (shown only for serum 3; empty triangle) HEK293 cells treated with secretin to increase surface expression of AQP1; the untreated and treated samples were tested in the usual RIPA for anti-AQP1 antibodies. The sera were also treated with AQP4-transfected HEK293 cells (shown only for serum 3; open circle). Serum 4 was also treated with an extract of AQP1-transfected HEK293 cells (open square).</p