第925回千葉医学会例会・第20回千葉大学医学部放射線医学教室同門会例会

Teriflunomide does not affect cell death in cultured primary microglia. Isolated microglia were treated with LPS (100 ng/ml) and different concentrations of teriflunomide (0.25–5 μM) for 12, 48, or 72 h followed by staining with propidium iodide (PI). (A, B, C) The percentage of gated PI+ CD11b/c+ cells is presented as mean + SD (n = 4). (TIF 718 kb

Influence of DMF and MMF on NO production of LPS stimulated microglia.

<p>After stimulation for 24 h with 10 ng/ml LPS a dose dependent decrease of NO production in cells incubated with DMF (5, 10, 50µM/ml) could be observed. All data represent three independent experiments compared to a daily standard curve using known concentrations of sodium nitrite in culture medium. Results are shown as means ± SEM. Significant post hoc effects versus controls (treated with LPS) are indicated by asterisks (*p<0.05, **p<0.01, ***p<0.001). CO: medium control without LPS.</p

Induction of apoptosis in mouse PBMC by DMF or MMF for 48h as determined with PI (A) and Annexin (B) staining by flow cytometry.

<p>For controls PBMC were treated with 1.0% methanol or with medium alone (CO). Results are shown as means ± SEM. Significant post hoc effects versus controls (treated with methanol) are indicated by asterisks (**p<0.01, ***p<0.001).</p

Additional file 3: Figure S1. of In vitro evaluation of physiologically relevant concentrations of teriflunomide on activation and proliferation of primary rodent microglia

Teriflunomide inhibits rodent T cell proliferation. Single-cell suspensions from spleens of adult Sprague-Dawley rats (Crl:CD) were prepared in complete IMDM medium. Freshly isolated rat CD4+ T cells were labeled with 2.5 μM CFSE, stimulated with plate-bound anti-CD3/CD28 mAb and treated with different concentrations of teriflunomide (0.25–5 μM) for 65–72 h. Control presents unstimulated CFSE-labeled cells. (A) Total number of T cells as shown in histograms for 0.5 μM teriflunomide was determined by flow cytometry. (B) For comparison of proliferation, the number of proliferating T cells (n = 4) repeated measures ANOVA with Bonferroni’s multiple comparison t test were used. Significant effects are indicated by asterisks (***p < 0.001). (TIF 162 kb

Influence of DMF and MMF on de- and remyelination in the cortex.

<p>Cortical myelin was demonstrated by scoring of MBP (A) and PLP (B). In the cortex score of 0 represents complete myelin protein loss, score of 4 represents normal myelin protein amount. Results are shown as means ± SEM.</p

Influence of DMF and MMF on glial reactions during de- and remyelination.

<p>Graphs represent cell numbers of Nogo-A (A), Olig-2 (B), and Mac-3 (D) positive cells in the corpus callosum. In C results of the APP staining to demonstrate axonal damage during cuprizone treatment are shown. Cell numbers are given as cells/mm². Results are shown as means ± SEM.</p

Additional file 2: of Effectors of Th1 and Th17 cells act on astrocytes and augment their neuroinflammatory properties

Clinical course of WT and α4−/− mice. EAE severity was monitored in WT and α4−/− mice following immunization with MOG35–55 in CFA. WT mice showed the signs of classical EAE whereas α4−/− mice had an ataxic EAE syndrome. Data is represented as mean clinical score + SEM (n = 6). (TIFF 419 kb

Additional file 4: of Effectors of Th1 and Th17 cells act on astrocytes and augment their neuroinflammatory properties

ELISA of astrocyte culture supernatants. Concentration of CCL2, CCL20, and IL-6 was measured in the culture supernatants of astrocytes cultured in medium, Th1 and Th17 supernatants. Since we did not remove T cells factors, to exclude any background resulting from T cells, supernatants used in these experiments to stimulate astrocytes were also tested for these factors. CCL2 and IL-6 was not detected in T cell supernatants whereas little amount of CCL20 was detected in Th17 supernatants. Actual concentration of CCL20 released by astrocytes was calculated by subtracting the concentrations obtained for Th17 supernatants. The results are mean ± SEM (n = 4) with *p ≤ 0.05 and **p ≤ 0.01. (TIFF 216 kb

Additional file 5: of Effectors of Th1 and Th17 cells act on astrocytes and augment their neuroinflammatory properties

Inhibition of reactive astrocytosis affects Th17 infiltration into the CNS. Expression of Rorc mRNA was measured in the spinal cord of wild-type (WT) or GFAP TK (Tg) mice with established EAE where at day 7 following onset of clinical symptoms the mice were either challenge with PBS (controls) or ganciclovir (GCV) to deplete astrocytes. The basal expression was adjusted to WT-PBS controls and fold changes in Rorc mRNA expression was determined for the other groups. Each point represents data from one mouse. Here we used Mann-Whitney test. (TIFF 276 kb

Effects of Murine and Human Bone Marrow-Derived Mesenchymal Stem Cells on Cuprizone Induced Demyelination

<div><p>For the treatment of patients with multiple sclerosis there are no regenerative approaches to enhance remyelination. Mesenchymal stem cells (MSC) have been proposed to exert such regenerative functions. Intravenous administration of human MSC reduced the clinical severity of experimental autoimmune encephalomyelitis (EAE), an animal model mimicking some aspects of multiple sclerosis. However, it is not clear if this effect was achieved by systemic immunomodulation or if there is an active neuroregeneration in the central nervous system (CNS). In order to investigate remyelination and regeneration in the CNS we analysed the effects of intravenously and intranasally applied murine and human bone marrow-derived MSC on cuprizone induced demyelination, a toxic animal model which allows analysis of remyelination without the influence of the peripheral immune system. In contrast to EAE no effects of MSC on de- and remyelination and glial cell reactions were found. In addition, neither murine nor human MSC entered the lesions in the CNS in this toxic model. In conclusion, MSC are not directed into CNS lesions in the cuprizone model where the blood-brain-barrier is intact and thus cannot provide support for regenerative processes.</p></div