Natalizumab treatment reduces L-selectin (CD62L) in CD4+ T cells

Background: The purpose of this research was to validate the low expression of L-selectin (CD62L) in natalizumab (NTZ)-treated patients. CD62L is involved in rolling and transmigration of leukocyte cells. A correlation between CD62LCD4+ T cells low expression and progressive multifocal leukoencephalopathy (PML) development has been suggested in multiple sclerosis (MS) patients treated with NTZ. Methods: We performed a flow cytometric analysis on peripheral blood mononuclear cells (PBMC); we collected from 23 healthy donors and 225 MS patients: untreated (n = 19) or treated with NTZ (n = 113), interferon-beta (n = 26), glatiramer acetate (n = 26), fingolimod (n = 23) and rituximab (n = 18). We have also analysed two PML/IRIS (immune reconstitution inflammatory syndrome) patients and four longitudinal samples of a NTZ-treated patients before and during the development of a clinical asymptomatic magnetic resonance imaging (MRI) lesion confirmed as PML by cerebrospinal fluid (CSF) examination. Thirty-five NTZ-treated patients were studied longitudinally with three samples taken 4 months apart. Results: The NTZ-treated patients showed a lower percentage of CD62L (33.68 %, n = 113) than first-line treated patients (44.24 %, n = 52, p = 0.0004). NTZ effect was already clear during the first year of treatment (34.68 %; p = 0.0184); it persisted in the following years and disappeared after drug withdrawal (44.08 %). Three percent of longitudinally analysed patients showed a percentage of CD62LCD4+ T cells under a hypothetical threshold and one patient with asymptomatic PML belongs to a group which expressed low percentage of CD62LCD4+ T cells. Conclusions: Our research confirms that NTZ has a specific effect on CD62LCD4+ T cells consisting in decreasing of the number of positive cells. The low level of CD62L found in a clinically asymptomatic PML patient strengthens its potential usefulness as a biomarker of high PML risk in NTZ-treated patients. A larger study is required to better confirm the data

Vitamin D binding protein isoforms and apolipoprotein E in cerebrospinal fluid as prognostic biomarkers of multiple sclerosis

Multiple sclerosis (MS) is a multifactorial autoimmune disease of the central nervous system with a heterogeneous and unpredictable course. To date there are no prognostic biomarkers even if they would be extremely useful for early patient intervention with personalized therapies. In this context, the analysis of inter-individual differences in cerebrospinal fluid (CSF) proteome may lead to the discovery of biological markers that are able to distinguish the various clinical forms at diagnosis.To this aim, a two dimensional electrophoresis (2-DE) study was carried out on individual CSF samples from 24 untreated women who underwent lumbar puncture (LP) for suspected MS. The patients were clinically monitored for 5 years and then classified according to the degree of disease aggressiveness and the disease-modifying therapies prescribed during follow up.The hierarchical cluster analysis of 2-DE dataset revealed three protein spots which were identified by means of mass spectrometry as Apolipoprotein E (ApoE) and two isoforms of vitamin D binding protein (DBP). These three protein spots enabled us to subdivide the patients into subgroups correlated with clinical classification (MS aggressive forms identification: 80%). In particular, we observed an opposite trend of values for the two protein spots corresponding to different DBP isoforms suggesting a role of a post-translational modification rather than the total protein content in patient categorization.These findings proved to be very interesting and innovative and may be developed as new candidate prognostic biomarkers of MS aggressiveness, if confirmed

Role of anti-osteopontin antibodies in multiple sclerosis and experimental autoimmune encephalomyelitis

Osteopontin (OPN) is highly expressed in demyelinating lesions in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). OPN is cleaved by thrombin into N- (OPN-N) and C-terminal (OPN-C) fragments with different ligands and functions. In EAE, administering recombinant OPN induces relapses, whereas treatment with anti-OPN antibodies ameliorates the disease. Anti-OPN autoantibodies (autoAbs) are spontaneously produced during EAE but have never been detected in MS. The aim of the study was to evaluate anti-OPN autoAbs in the serum of MS patients, correlate them with disease course, and recapitulate the human findings in EAE. We performed ELISA in the serum of 122 patients collected cross-sectionally, and 50 patients with relapsing-remitting (RR) disease collected at diagnosis and followed longitudinally for 10 years. In the cross-sectional patients, the autoAb levels were higher in the RR patients than in the primary- and secondary-progressive MS and healthy control groups, and they were highest in the initial stages of the disease. In the longitudinal group, the levels at diagnosis directly correlated with the number of relapses during the following 10 years. Moreover, in patients with active disease, who underwent disease-modifying treatments, autoAbs were higher than in untreated patients and were associated with low MS severity score. The autoAb displayed neutralizing activity and mainly recognized OPN-C rather than OPN-N. To confirm the clinical effect of these autoAbs in vivo, EAE was induced using myelin oligodendrocyte glycoprotein MOG35-55 in C57BL/6 mice pre-vaccinated with ovalbumin (OVA)-linked OPN or OVA alone. We then evaluated the titer of antibodies to OPN, the clinical scores and in vitro cytokine secretion by spleen lymphocytes. Vaccination significantly induced antibodies against OPN during EAE, decreased disease severity, and the protective effect was correlated with decreased T cell secretion of interleukin 17 and interferon-\u3b3 ex vivo. The best effect was obtained with OPN-C, which induced significantly faster and more complete remission than other OPN vaccines. In conclusion, these data suggest that production of anti-OPN autoAbs may favor remission in both MS and EAE. Novel strategies boosting their levels, such as vaccination or passive immunization, may be proposed as a future strategy in personalized MS therapy

Additional file 1: Figure S1 of Natalizumab treatment reduces L-selectin (CD62L) in CD4+ T cells

Set up procedure. A Expression of CD4+ T cells. PBMCs were analysed before (solid symbol) and after (open symbol) freezing in group A (six patients, four HD and two first-line treated MS; squares) and NTZ (five patients; diamonds). B Expression of CD62L on CD4+ T cells. PBMCs were analysed before (solid symbol) and after (open symbol) freezing in group A (six patients, four HD and two first-line treated MS; squares) and NTZ (five patients, diamonds). After freezing CD62L expression decreased by 31 % in the CTRL group and by 33 % in the NTZ group. C Analysis of stability and reproducibility. The frozen samples from the same patient were evaluated for CD62L expression in two different work sessions 3 months apart. The statistical differences were calculated with Student's paired t test

Statistical analysis of spots differently expressed between cluster A and cluster B.

<p>§ In the fold change column cluster B is compared to cluster A. Values are expressed as the mean of Vol% ± SD of spots detected in 2-DE map from each patient included in cluster A or B.</p><p>NS = not statistical significant (Mann Whitney test).</p><p>Statistical analysis of spots differently expressed between cluster A and cluster B.</p

Three spot-based cluster analysis (ID 288, ID 289 and ID469).

<p>(A) Dendrogram of the hierarchical cluster analysis based on the %Vol values of spots ID288, ID289 (DBP isoforms) and ID469 (ApoE), showing wider inter-individual range of %Vol values in the differently expressed spots. (B) Column bar graph showing %Vol values of spots ID 288, ID 289, ID 469 in clusters B1, C1 and D1. Values are expressed as the mean of the technical replicates ± SD (bars).</p

Biochemical features of CSF in individual patients enrolled in the study.

<p>Biochemical features of CSF in individual patients enrolled in the study.</p

Cluster analysis based on 2-DE data from CSF gels and differently expressed spots.

<p>(A) Hierarchical cluster analysis based on the %Vol values of the 239 most representative spots of the analysed gel population. For each spot, the mean value of %Vol was calculated considering the best two replicates (average SD lower than 30%) among 2-DE maps performed in triplicates. Two main clusters (A and B) and five outliers were identified. (B) A representative 2-DE map of CSF: differently expressed spots between cluster A and B are outlined in black.</p

Two spot-based cluster analysis (spot ID 288 e ID 289).

<p>(A) Graph showing the results of linear discriminant analysis based on the two linear functions (f1 = 33.813*(%Vol spot ID288) + 48.942*(%Vol spot ID289)– 7.155; f2 = 103.508*(%Vol spot ID288) + 42.871*(%Vol spot ID289)– 15.248) allowing optimal separation of clusters G and Z based on the measured variables (spot ID 288 and ID 289). The mathematical function separating the two groups is also reported. (B) Hierarchical cluster analysis based on the %Vol values of spots ID288 and ID289 identify two clusters, G and Z, and one outlier MS48. The clinical classification of the patients included in the study is indicated alongside the sample number: L = MS patients with a mildly aggressive disease; LB = MS patients with a benign form of the disease; M = MS patients with a moderately aggressive disease; H = MS patients with a highly aggressive disease; PP = MS patients with a primary progressive form of the disease; NA: patients lost to follow-up; no MS: patient with an unconfirmed diagnosis of MS. (C) Column bar graph shows %Vol values of spots ID288 and ID289 in clusters Z and G: values are expressed as the mean of three technical replicates ± SD (bars). * <i>P</i><0.05, *** <i>P</i><0.001 (Mann Whitney’s Test). (D) Spearman statistical analysis shows a significant inverse correlation between spots ID288 and ID289%Vol values for each subject (r = -0.49, p = 0.015).</p

%Vol of spots ID288, ID289 and ID469 in individual patients.

<p>Graphical representation of %Vol values of spots ID288, ID289 and ID469 showing the widest inter-individual range in the population studied (0,00–0,44; 0,06–0,33; 0,01–0,54 respectively). The %Vol values are expressed as the mean of at least three replicates ± SD.</p