MSJ765666_supplementary_figure_1 – Supplemental material for Serum neurofilament light chain is a biomarker of acute and chronic neuronal damage in early multiple sclerosis

<p>Supplemental material, MSJ765666_supplementary_figure_1 for Serum neurofilament light chain is a biomarker of acute and chronic neuronal damage in early multiple sclerosis by Nelly Siller, Jens Kuhle, Muthuraman Muthuraman, Christian Barro, Timo Uphaus, Sergiu Groppa, Ludwig Kappos, Frauke Zipp and Stefan Bittner in Multiple Sclerosis Journal</p

CNS invasion of neuroantigen-specific T cells is impaired by ceftriaxone.

<p>Splenocytes from TCR-transgenic 2D2 mice were stimulated for 5 days with MOG peptide (20 µg/ml) in the presence or absence of 500 µM ceftriaxone <i>in vitro</i> and adoptively transferred into WT C57BL/6 mice (3×10⁶ splenocytes/mice) pre-treated for 5 days with or without ceftriaxone (200 mg/kg i.p.). Dot plot show numbers of CNS invasive CD4⁺ T cells analysed 4 days after transfer using whole-brain FACS analysis. Mean absolute numbers of T cells/brain calculated from 3 to 4 mice pooled per experimental group are indicated in each histogram.</p

Clinical parameters of the EAE course.

<p>Disease incidence was 100% in all experimental groups of the experiments shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003149#pone-0003149-g001" target="_blank">Fig. 1 (white)</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003149#pone-0003149-g005" target="_blank">Fig. 5 (grey)</a>. CTX denotes ceftriaxone, DHK denotes dihydrokainate.</p

A β-lactam antibiotic profoundly attenuates the clinical course of MOG-induced EAE in mice.

<p>(A) Time course of neurological symptoms after immunization of WT C57BL/6 mice with a MOG peptide (MOG_35–55). Mice were treated with ceftriaxone (200 mg/kg/d i.p.) either from the day of immunization (MOG+CTX permanent; filled squares) or from the individual onset of symptoms (MOG+CTX therapeutic; empty circles). MOG-immunized control mice (MOG) were injected with an equivalent volume of saline (MOG; filled circles). The degree of neurological impairment was assessed using a 10-point scoring system. (B) Mean cumulative score of MOG immunized mice treated with saline (control; n = 8) and with ceftriaxone from the day of immunization (permanent; n = 8) or from the individual onset of symptoms (therapeutical; n = 8). Differences between the 3 experimental groups are significant (control vs. permanent: p<0.001 ***; control vs. therapeutical: p = 0.05 *; permanent vs. therapeutical: p<0.01 **).</p

Ceftriaxone attenuates the clinical EAE course in mice in the presence of the EAAT2 transport inhibitor dihydrokainate.

<p>(A) Time course of neurological symptoms after immunization of WT C57BL/6 mice with human recombinant MOG. Mice were treated from the day of immunization with ceftriaxone alone (MOG+CTX; filled triangles; 200 mg/kg/d i.p.) or in combination with dihydrokainate (MOG+CTX+DHK; 10 mg/kg/d i.p.; empty triangles). MOG-immunized control mice were injected with an equivalent volume of saline alone (MOG; filled circles) or together with dihydrokainate (MOG+DHK; empty circles). The degree of neurological symptoms was assessed using a 10-point scoring system. (B) Mean cumulative score of mice from the different experimental groups. Differences between the experimental groups are significant (MOG vs. MOG+ceftriaxone: p = 0.004 **; MOG vs. MOG+ceftriaxone+dihydrokainate: p = 0.004 **; MOG vs. MOG+DHK: p = 0.05 *).</p

Reduced T cell response is due to ceftriaxone-induced modulation of cellular antigen-presentation.

<p>(A) Ceftriaxone concentration-dependence of CD3/CD28 stimulation induced proliferation of murine CD4⁺ T cells. Ceftriaxone does not inhibit [3H]thymidine incorporation in T cells (p([ceftriaxone] = 0 µM vs. [ceftriaxone] = 500 µM) = 0.12; n = 6 respectively). (B) Proliferation of murine CD4⁺ T cells (TCs) cocultured with dendritic cells (DCs) previously loaded with MOG peptide (50 µg/ml) in the absence and presence of different ceftriaxone concentrations. MOG-preincubation of dendritic cells in the presence of ceftriaxone impaired subsequent proliferation of T cells (p([ceftriaxone] = 0 µM vs. [ceftriaxone] = 500 µM): p = 0.05 *; n = 6). (C) Ceftriaxone concentration dependence of supernatant IFNγ and IL17 levels from the experiment described in (B). MOG-preincubation of dendritic cells in the presence of ceftriaxone lowered IFNγ and IL17 levels in a concentration dependent manner (p([ceftriaxone] = 0 µM vs. [ceftriaxone] = 500 µM): IFNγ: p<0.001 ***, IL17: p<0.001 ***; n = 6 respectively).</p

Dihydrokainate-sensitive radioactive glutamate uptake in a rat primary mixed glial cell culture is not influenced by ceftriaxone.

<p>(A) [3H]-glutamate (60 µM) uptake was measured in a rat primary mixed glial cell culture after 5 day of incubation with (white bars) or without (black bars) 10 µM ceftriaxone using either a NaCl-based external solution in the absence (left) or the presence (right) of 1 mM dihydrokainate or using a sodium-free NMDG-Cl-based external solution (middle). Substitution of external sodium by NMDG significantly reduced the uptake in the absence as well as in the presence of ceftriaxone (0.22±0.02 and 0.27±0.02, respectively; n = 3 trials consisting of 2 samples each; p<0.001 ***). Dihydrokainate lowered glutamate uptake to 0.47±0.03 in the absence and 0.55±0.08 in the presence of ceftriaxone (n = 3 trials consisting of 2 samples each; p<0.001 ***). (B) A ceftriaxone concentration-dependence of the [3H]-glutamate uptake could not be observed after 5 days of incubation with concentrations between 0 and 500 µM (p([ceftriaxone] = 0 µM vs. [ceftriaxone] = 500 µM) = 0.19; n = 3 trials consisting of 6 samples).</p

Ceftriaxone does not modulate phenotypical but functional properties of peripheral immune cells.

<p>(A) FACS-analysis of the activation markers CD40 (upper left), CD80 (upper right), CD86 (lower left) and MHCII (lower right) on CD11b⁺ CD11c⁺ APCs from spleen of untreated (thick lines) and ceftriaxone-treated (thin lines) MOG-immunized mice at the disease maximum. Geometric mean fluorescence intensities of all marker were similar between experimental groups. (B) Immunophenotyping of CD4⁺ (upper panels) and CD8⁺ (lower panels) T cells from spleen of non-immunized (left panels) as well as untreated (middle) and ceftriaxone-treated (right) MOG-immunized mice. Relative fractions of T cells as assed by CD40 and CD62L expression were similar between experimental groups. (C, D) MOG-recall experiments performed with splenocytes from untreated as well as permanently and therapeutically treated MOG-immunized mice at the disease maximum (C) and in the residual state (D) in the total absence of ceftriaxone <i>in vitro</i>. MOG-specific supernatant IFNγ-levels were significantly reduced relative to antigen-independent CD3/CD28 bead-stimulation in samples from MOG-immunized mice treated with ceftriaxone as compared to untreated MOG-immunized mice at the disease maximum (p (permanent) = 0.02 *; p (therapeutical)<0.01 **) and the residual state (p (permanent)<0.01 **; p (therapeutical)<0.01 **; n = 3 samples out of 3 animals, respectively). There was no difference whether mice were treated permanently or only after disease onset.</p

Additional file 1: of ALAIN01—Alemtuzumab in autoimmune inflammatory neurodegeneration: mechanisms of action and neuroprotective potential

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Additional file 1 of Assessment of technical and clinical utility of a bead-based flow cytometry platform for multiparametric phenotyping of CNS-derived extracellular vesicles

Additional file 1: Table S1. Capture bead composition of the MACSPlex EV Kit Neuro, human, panel A and B. Figure S1. Workflow and gating strategy for flow cytometry analysis of EV Neuro panel A and B. (a) Capture beads (CB) are incubated with isolated EVs. After washing, detection antibody cocktail (DA) containing APC-labelled antibodies against CD9, CD63, and CD81 are added. After incubation and washing, flow cytometry (FC) measurement is performed. (b) Gating strategy for flow cytometry assessment of signal intensities for each capture bead population in EV Neuro panel A and B. Figure S2. Number of detected events and background signals. (a) Background median signal intensities for each capture bead population (CB) after incubation with detection antibody cocktail (DA). Data was obtained from 10 individual measurements of MACSPlex buffer (MPB) with CB and anti-CD9/anti-CD63/anti-CD81-APC DA. Medians are represented as bars. (b) Number of detected barcoded beads used for estimation of median signal intensities (upper bold horizontal line: 50 events, lower bold horizontal line: 10 events). Note: While for most of the targets the number of detected events was consistently between 200 and 500, for BDNF, CD24, and CD56 it was repeatedly below 50 events​ (for BDNF several times below 10)​. This results in a small number of events considered to estimate the median signal intensities, which might hamper reproducibility of obtained data for these targets. Figure S3. Titration of EVs derived from glioblastoma cells – relative profiles. Relative signal intensities achieved with different inputs of NCH82 EVs (a) and LN18 EVs (b) corresponding to Fig. 3 (a) and (b), respectively. The graphs illustrate that despite different EV input, the EV profile remains stable for a specific cell line. Figure S4. Phenotyping of serum EVs from GB patients and healthy controls. (a) EV Neuro signal intensities for EVs separated from the serum of three GB patients by either SEC or combination of SEC and UC (SEC-UC). (b) Relative signal intensities as signal of target divided by the total signal of all markers (in %) of (a). Bars represent mean values. The graphs show that appending UC to SEC leads to increased signal intensity with a constant EV profile. (c) EV Neuro signal intensities for EVs separated from the serum of GB patients or healthy controls by SEC-UC. Corresponding to Fig. 4 in the main text. Note that different input volumes were tested in the GB and HC group, thus the means are not comparable. The graph illustrates the high variation of signal intensities between the individuals of a group and the absence of specific markers unique for the GB condition (or vice versa). Figure S5. EV Neuro signal intensities for EVs separated from the serum of MAD patients by CD81-immunoaffinity capture. Corresponding to Fig. 7a in the main text. Figure S6. Heatmap clustering analysis of all blood-derived EV samples. (a) Heatmap and clustering analysis of all plasma and serum samples analyzed in this study. Signal intensities were normalized to the signal of CD9 (NSI). The genuine EV marker CD9 was used for normalization since it was not affected by the isolation technique and appeared quite stable throughout all samples analyzed. BCL = lab of blood collection, BCT = type of blood collection