Additional file 1: Figure S1. of Early strong intrathecal inflammation in cerebellar type multiple system atrophy by cerebrospinal fluid cytokine/chemokine profiles: a case control study

A schematic drawing indicating measurement of each part of the hindbrain. 1. Vertical diameter of vermis. 2. Anteroposterior diameter of vermis. 3. Anteroposterior diameter of pontine base. 4. Anteroposterior diameter of medulla oblongata. Figure S2. Clustering of correlations between each CSF cytokine level in SCA patients using the same order of cytokines as in MSA-C. Color codes indicate R values of correlations calculated using Pearson’s correlation coefficient. * p < 0.05, ** p < 0.01. CSF: cerebrospinal fluid, SCA: spinocerebellar ataxia, MSA-C: multiple system atrophy cerebellar-type. Figure S3. Heatmap analysis of correlations between cytokine levels and disease duration in SCA patients. R values of Pearson’s correlation coefficient analysis are divided into quintiles, and p-values calculated using one-way ANOVA are indicated as a heatmap. There were no significant correlations between cytokines and disease duration in SCA patients. SCA: spinocerebellar ataxia. Figure S4. Heatmap analysis of correlations between cytokine levels and MRI measurement in SCA. R-values of Pearson’s correlation coefficient analysis are divided into quintiles, and p-values calculated using one-way ANOVA are indicated as a heatmap. Among the 27 cytokines studied, only PDGF, IL-12(p70), GM-CSF, and MIP-1α showed significant negative correlations with MRI measurements. GM-CSF: granulocyte-macrophage colony-stimulating factor, IL: interleukin, MIP: macrophage inflammatory protein, MRI: magnetic resonance imaging, PDGF: platelet-derived growth factor, SCA: spinocerebellar ataxia. Table S1. Detection rates of cytokines/chemokines and growth factors in cerebrospinal fluid. Table S2. Summary of dysregulated cerebrospinal fluid cytokines (PDF 188 kb

Correlation of cytokine and chemokine levels in patients with RRMS and NMO/NMOSD in the relapse phase.

<p>Among the cytokines and chemokines analyzed, distances of each pair of cytokines/chemokines, based on Spearman's correlation coefficient, of RRMS and NMO/NMOSD in the relapse phase were shown as a heatmap. NMO = neuromyelitis optica; NMOSD = neuromyelitis optica spectrum disorder; RRMS = relapsing remitting multiple sclerosis.</p

Demographic features of patients.

<p>NA = not applicable; NMO = neuromyelitis optica; NMOSD = neuromyelitis optica spectrum disorder; OND = other non-inflammatory neurological diseases; PPMS = primary progressive multiple sclerosis; RRMS = relapsing remitting multiple sclerosis; CSF = cerebrospinal fluid; SD = standard deviation; EDSS = Expanded Disability Status Scale.</p>*, #<p>p<0.05.</p

Correlations of cytokines/chemokines in RRMS and NMO/NMOSD in the relapse phase.

<p>NMO = neuromyelitis optica; NMOSD = neuromyelitis optica spectrum disorder; OND = other non-inflammatory neurological diseases; RRMS = relapsing remitting multiple sclerosis. rho = Spearman's correlation coefficient, <i>^corrp</i> = corrected p value by Benjamini-Hochberg method.</p

image_1.PDF

<p>We recently reported that deletion-type copy number variations of the T cell receptor (TCR) γ, α, and δ genes greatly enhanced susceptibility to multiple sclerosis (MS). However, the effect of abnormal TCR γδ gene rearrangement on MS pathogenesis remains unknown. In the present study, we aimed to clarify γδ TCR repertoire alterations and their relationship to clinical and immunological parameters in MS patients by comprehensive flow cytometric immunophenotyping. Peripheral blood mononuclear cells obtained from 30 untreated MS patients in remission and 23 age- and sex-matched healthy controls (HCs) were stained for surface markers and intracellular cytokines after stimulation with phorbol 12-myristate 13-acetate and ionomycin, and analyzed by flow cytometry. MS patients showed significantly decreased percentages of Vδ2⁺ and Vδ2⁺Vγ9⁺ cells in γδ T cells (p^corr = 0.0297 and p^corr = 0.0288, respectively) and elevated Vδ1/Vδ2 ratios compared with HCs (p = 0.0033). The percentages of interferon (IFN)-γ⁺Vδ2⁺ and interleukin (IL)-17A⁺IFN-γ⁺Vδ2⁺ cells in γδ T cells, as well as IFN-γ⁺ cells in Vδ2⁺ γδ T cells, were significantly lower in MS patients than in HCs (p^corr < 0.0009, p^corr = 0.0135, and p^corr = 0.0054, respectively). The percentages of Vδ2⁺ and Vδ2⁺Vγ9⁺ cells in γδ T cells were negatively correlated with both the Expanded Disability Status Scale score (r = −0.5006, p = 0.0048; and r = −0.5040, p = 0.0045, respectively) and Multiple Sclerosis Severity Score (r = –0.4682, p = 0.0091; and r = –0.4706, p = 0.0087, respectively), but not with age at disease onset, disease duration, or annualized relapse rate. In HCs, the percentages of Vδ2⁺ and Vδ2⁺Vγ9⁺ cells of total CD3⁺ T cells had strong positive correlations with the percentage of CD25⁺CD127^low/− cells in CD4⁺ T cells (r = 0.7826, p < 0.0001; and r = 0.7848, p < 0.0001, respectively), whereas such correlations were totally absent in MS patients. These findings suggest that decreased Vδ2⁺Vγ9⁺ γδ T cells are associated with disability in MS. Therefore, the Vδ1/Vδ2 ratio might be a candidate biomarker for predicting disease severity in MS.</p

table_1.docx

<p>We recently reported that deletion-type copy number variations of the T cell receptor (TCR) γ, α, and δ genes greatly enhanced susceptibility to multiple sclerosis (MS). However, the effect of abnormal TCR γδ gene rearrangement on MS pathogenesis remains unknown. In the present study, we aimed to clarify γδ TCR repertoire alterations and their relationship to clinical and immunological parameters in MS patients by comprehensive flow cytometric immunophenotyping. Peripheral blood mononuclear cells obtained from 30 untreated MS patients in remission and 23 age- and sex-matched healthy controls (HCs) were stained for surface markers and intracellular cytokines after stimulation with phorbol 12-myristate 13-acetate and ionomycin, and analyzed by flow cytometry. MS patients showed significantly decreased percentages of Vδ2⁺ and Vδ2⁺Vγ9⁺ cells in γδ T cells (p^corr = 0.0297 and p^corr = 0.0288, respectively) and elevated Vδ1/Vδ2 ratios compared with HCs (p = 0.0033). The percentages of interferon (IFN)-γ⁺Vδ2⁺ and interleukin (IL)-17A⁺IFN-γ⁺Vδ2⁺ cells in γδ T cells, as well as IFN-γ⁺ cells in Vδ2⁺ γδ T cells, were significantly lower in MS patients than in HCs (p^corr < 0.0009, p^corr = 0.0135, and p^corr = 0.0054, respectively). The percentages of Vδ2⁺ and Vδ2⁺Vγ9⁺ cells in γδ T cells were negatively correlated with both the Expanded Disability Status Scale score (r = −0.5006, p = 0.0048; and r = −0.5040, p = 0.0045, respectively) and Multiple Sclerosis Severity Score (r = –0.4682, p = 0.0091; and r = –0.4706, p = 0.0087, respectively), but not with age at disease onset, disease duration, or annualized relapse rate. In HCs, the percentages of Vδ2⁺ and Vδ2⁺Vγ9⁺ cells of total CD3⁺ T cells had strong positive correlations with the percentage of CD25⁺CD127^low/− cells in CD4⁺ T cells (r = 0.7826, p < 0.0001; and r = 0.7848, p < 0.0001, respectively), whereas such correlations were totally absent in MS patients. These findings suggest that decreased Vδ2⁺Vγ9⁺ γδ T cells are associated with disability in MS. Therefore, the Vδ1/Vδ2 ratio might be a candidate biomarker for predicting disease severity in MS.</p

table_2.docx

<p>We recently reported that deletion-type copy number variations of the T cell receptor (TCR) γ, α, and δ genes greatly enhanced susceptibility to multiple sclerosis (MS). However, the effect of abnormal TCR γδ gene rearrangement on MS pathogenesis remains unknown. In the present study, we aimed to clarify γδ TCR repertoire alterations and their relationship to clinical and immunological parameters in MS patients by comprehensive flow cytometric immunophenotyping. Peripheral blood mononuclear cells obtained from 30 untreated MS patients in remission and 23 age- and sex-matched healthy controls (HCs) were stained for surface markers and intracellular cytokines after stimulation with phorbol 12-myristate 13-acetate and ionomycin, and analyzed by flow cytometry. MS patients showed significantly decreased percentages of Vδ2⁺ and Vδ2⁺Vγ9⁺ cells in γδ T cells (p^corr = 0.0297 and p^corr = 0.0288, respectively) and elevated Vδ1/Vδ2 ratios compared with HCs (p = 0.0033). The percentages of interferon (IFN)-γ⁺Vδ2⁺ and interleukin (IL)-17A⁺IFN-γ⁺Vδ2⁺ cells in γδ T cells, as well as IFN-γ⁺ cells in Vδ2⁺ γδ T cells, were significantly lower in MS patients than in HCs (p^corr < 0.0009, p^corr = 0.0135, and p^corr = 0.0054, respectively). The percentages of Vδ2⁺ and Vδ2⁺Vγ9⁺ cells in γδ T cells were negatively correlated with both the Expanded Disability Status Scale score (r = −0.5006, p = 0.0048; and r = −0.5040, p = 0.0045, respectively) and Multiple Sclerosis Severity Score (r = –0.4682, p = 0.0091; and r = –0.4706, p = 0.0087, respectively), but not with age at disease onset, disease duration, or annualized relapse rate. In HCs, the percentages of Vδ2⁺ and Vδ2⁺Vγ9⁺ cells of total CD3⁺ T cells had strong positive correlations with the percentage of CD25⁺CD127^low/− cells in CD4⁺ T cells (r = 0.7826, p < 0.0001; and r = 0.7848, p < 0.0001, respectively), whereas such correlations were totally absent in MS patients. These findings suggest that decreased Vδ2⁺Vγ9⁺ γδ T cells are associated with disability in MS. Therefore, the Vδ1/Vδ2 ratio might be a candidate biomarker for predicting disease severity in MS.</p

The relapsed patients have greater percentages of CD4⁺TCM at 3 and 6 months.

<p>Comparisons of TCM (A) and TEM (B) percentages in CD4⁺T cells at pre-treatment (MS PT) and the indicated periods of fingolimod treatment between MS patients with (open diamonds) and without (closed circles) relapse during the therapy. Box-whisker plots are shown. W = week; M = month.</p

Connexin 43 Astrocytopathy Linked to Rapidly Progressive Multiple Sclerosis and Neuromyelitis Optica

<div><p>Background</p><p>Multiple sclerosis (MS) and neuromyelitis optica (NMO) occasionally have an extremely aggressive and debilitating disease course; however, its molecular basis is unknown. This study aimed to determine a relationship between connexin (Cx) pathology and disease aggressiveness in Asian patients with MS and NMO.</p> <p>Methods/Principal Findings</p><p>Samples included 11 autopsied cases with NMO and NMO spectrum disorder (NMOSD), six with MS, and 20 with other neurological diseases (OND). Methods of analysis included immunohistochemical expression of astrocytic Cx43/Cx30, oligodendrocytic Cx47/Cx32 relative to AQP4 and other astrocytic and oligodendrocytic proteins, extent of demyelination, the vasculocentric deposition of complement and immunoglobulin, and lesion staging by CD68 staining for macrophages. Lesions were classified as actively demyelinating (n=59), chronic active (n=58) and chronic inactive (n=23). Sera from 120 subjects including 30 MS, 30 NMO, 40 OND and 20 healthy controls were examined for anti-Cx43 antibody by cell-based assay. Six NMO/NMOSD and three MS cases showed preferential loss of astrocytic Cx43 beyond the demyelinated areas in actively demyelinating and chronic active lesions, where heterotypic Cx43/Cx47 astrocyte oligodendrocyte gap junctions were extensively lost. Cx43 loss was significantly associated with a rapidly progressive disease course as six of nine cases with Cx43 loss, but none of eight cases without Cx43 loss regardless of disease phenotype, died within two years after disease onset (66.7% vs. 0%, <i>P</i>=0.0090). Overall, five of nine cases with Cx43 loss and none of eight cases without Cx43 loss had distal oligodendrogliopathy characterized by selective myelin associated glycoprotein loss (55.6% vs. 0.0%, <i>P</i>=0.0296). Loss of oligodendrocytic Cx32 and Cx47 expression was observed in most active and chronic lesions from all MS and NMO/NMOSD cases. Cx43-specific antibodies were absent in NMO/NMOSD and MS patients.</p> <p>Conclusions</p><p>These findings suggest that autoantibody-independent astrocytic Cx43 loss may relate to disease aggressiveness and distal oligodendrogliopathy in both MS and NMO.</p> </div

Counts of lymphocytes, CD4⁺T, and CD8⁺T cells are decreased from 2 weeks.

<p>Effects of fingolimod on peripheral blood lymphocyte counts (A) and absolute numbers and percentages of CD4^<b>+</b>T (B) and CD8^<b>+</b>T (C) cells in lymphocytes of healthy controls (HCs) and MS patients at pre-treatment (MS PT) and the indicated periods of fingolimod treatment. The numbers examined at each time point were: HC = 18, and MS PT = 23, 2W = 20, 1M = 17, 2M = 19, 3M = 23, 6M = 20, 12M = 18. The horizontal bars indicate the mean values. W = week; M = month. ***<i>p</i><0.0001, *<i>p</i><0.05.</p