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Objective: Intrathecal Immunoglobulin M synthesis (IgM Intrathecal Fraction (IF) +) and spinal MRI lesions are both strong independent predictors of higher disease activity and severity in multiple sclerosis (MS). We investigated whether IgM IF + is associated with spinal cord manifestation and higher neuroaxonal damage in early MS. Methods: In 122 patients with a first demyelinating event associations between (1) spinal versus (vs) non-spinal clinical syndrome (2) spinal vs cerebral T2-weighted (T2w) and (3) contrast-enhancing (CE) lesion counts with IgG IF + (vs IgG IF −) or IgM IF + (vs IgM IF −) were investigated by logistic regression adjusted for age and sex, respectively. For serum neurofilament light chain (sNfL) analysis patients were categorized for presence or absence of oligoclonal IgG bands (OCGB), IgG IF and IgM IF (>0% vs 0%, respectively): (1) OCGB −/IgG IF −/IgM IF −; (2) OCGB +/IgG IF −/IgM IF −; (3) OCGB +/IgG IF +/IgM IF −; and (4) OCGB +/IgG IF +/IgM IF +. Associations between categories 2 to 4 vs category 1 with sNfL concentrations were analyzed by robust linear regression, adjusted for sex and MRI parameters. Results: Patients with a spinal syndrome had a 8.36-fold higher odds of IgM IF + (95%CI 3.03–23.03; p < 0.01). Each spinal T2w lesion (odds Ratio 1.39; 1.02–1.90; p = 0.037) and CE lesion (OR 2.73; 1.22–6.09; p = 0.014) was associated with an increased risk of IgM IF + (but not of IgG IF +); this was not the case for cerebral lesions. OCGB +/IgG IF +/IgM IF + category patients showed highest sNfL levels (estimate:1.80; 0.55–3.06; p < 0.01). Interpretation: Intrathecal IgM synthesis is strongly associated with spinal manifestation and independently more pronounced neuroaxonal injury in early MS, suggesting a distinct clinical phenotype and pathophysiology. ANN NEUROL 2022.

Additional file 1: Figure S1. MOG35-55-induced EAE clinical score. (A) MOG35–55-induced EAE in C57BL/6J mice by immunization with 200 μg of MOG35–55, emulsified in CFA on day 0. Mice also received 300ng of PTX i.v. on days 0 and 2. EAE disease severity was followed until chronic phase (d21) and mean clinical score ± SEM for n=10 mice/group were shown. (B) Percentage of disease incidence of immunized mice described in (A) is shown. (C) TNFα production by CD4+ T lymphocytes isolated at peak disease (d14) from spleen, LN and CNS after in vitro stimulation. Representative flow cytometric plots (left panels) and quantification (right panels) of CD4+c-Met- vs c-Met+ T cells are depicted. Mean values ± SEM for n=8 mice/group are shown. (D) Representative flow cytometric histograms (left panels) and Gmean/Isotype quantification (right panels) of CCR4 and PSGL-1 expression on CD4+c-Met- and c-Met+ T cells from spleen, LN and CNS extracted at peak disease (d14). Mean values ± SEM for n=5 mice/group are shown. Figure S2. 2D2 CD4+Vα3.2+c-Met- and c-Met+ polarized Th1 cells characterization in vitro. (A) Flow cytometry gating strategy for the identification of 2D2 CD4+Vα3.2+ T cells. Live lymphocytes were first selected for their morphology using FSC/SSC parameters, followed by the exclusion of doublets and dead cells (AQUA), and CD4+Vα3.2+ T lymphocytes were subsequently selected. (B) Representative flow cytometric histograms at day 6 post-Th1 differentiation of the expression of Itgα9 on 2D2 CD4+Vα3.2+c‐Met- and c‐Met+ T cells. (C) Gmean/Isotype quantification by flow cytometry of 2D2 polarized Th1 cells at day 6 post-differentiation of CCR4, CCR3 and PSGL-1 gated on 2D2 CD4+Vα3.2+c‐Met-and c‐Met+ T cells. Data are representative of three independent experiments and mean values ± SEM for n=3 mice/group are shown. Figure S3. Trafficking profiles of 2D2 c-Met+ polarized Th1 T cells on non-activated HUVECs in an in vitro flow assay system. (A) Representative flow cytometry plots for the normalization of the relative number of transmigrated cells (red population) using fluorescent counting beads (blue population) upon stimulation with indicated antibodies. (B) Adherent polarized 2D2 CD4+Vα3.2+c-Met- and c-Met+ Th1 T cells in coculture on non-activated HUVECs were individually tracked and monitored for transmigration between different compartments for 45 minutes. Captured (left panel), transmigrating (center panel) and transmigrated (right panel) T cells were analyzed separately. Data are presented as the means of 3 fields and are representative of three independents experiment. Mean values ± SD are shown. (C) Absolute numbers for c-Met- and c-Met+ captured (top panels) and transmigrated (bottom panels) cells in presence (left panels) or absence (right panels) of CXCL12 at indicated time points counted during the in vitro flow assay system described in Fig. 4C and D. Figure S4. Integrin and cytokine expression of Th1 cells from 2D2 Vα3.2+CD4CreItgα4fl/fl and Vα3.2+CD4Cre control littermate mice. (A) Spleen cells from control 2D2 Vα3.2+CD4Cre and 2D2 Vα3.2+CD4CreItgα4fl/fl mice were polarized in vitro into Th1 cells and the expression of Itgα4, Itgb1 (VLA4 second associated subunit) and ItgαL (LFA-1 integrin subunit) were assessed by flow cytometry. Data arerepresentative of three independent experiments and mean values ± SEM for n=5 mice/group are shown; ***p≤0.001 by unpaired two‐tailed Student's t‐test. (B) Th1 polarized cells were stimulated with PMA/ionomycin and analyzed for cytokine secretion by intracellular cytokines staining. Percentages of IFNγ and IL-17 expressing Th1 cells from control 2D2 Vα3.2+CD4Cre and 2D2 Vα3.2+CD4CreItgα4fl/fl mice were determined. Data are representative of three independent experiments and mean values ± SEM for n=5 mice/group are shown. Figure S5. Adoptive transfer of Itgα4-deficient 2D2 CD4+c-Met+ Th1 cells induces delayed and reduced EAE incidence, severity, and decreased infiltrated T cell quantifications. (A) Percentage of disease incidence of EAE induced mice described in Fig.5 are shown. (B) Survival curves of EAE induced mice described in Fig. 5 are shown. (C) Quantification of CNS CD4+Vα3.2+ infiltrating T cells by flow cytometry at day 18 post-Th1 transfer. Data are representative of 2 independent experiments and mean values ± SEM for n=3 mice/group are shown; **p≤0.01,***p≤0.001 by two-way ANOVA followed by Tukey’s post hoc test. (D) Representative flow cytometric plots of IFNγ and IL-17 production by Th1 infiltrating cells in the CNS at day 18 post-CD4+Vα3.2+ Th1 cells transfer and quantification of IFNγ-producing CD4+Vα3.2+ Th1 cells is shown on the right. Data are representative of 2 independent experiments and mean values ± SEM were shown for n=3 mice/group; *p≤0.05 by unpaired two‐tailed Student's t‐test. Table S1. Antibodies for flow cytometry

BACKGROUND Serum neurofilament light chain (sNfL) is a biomarker of neuronal damage that is used not only to monitor disease activity and response to drugs and to prognosticate disease course in people with multiple sclerosis on the group level. The absence of representative reference values to correct for physiological age-dependent increases in sNfL has limited the diagnostic use of this biomarker at an individual level. We aimed to assess the applicability of sNfL for identification of people at risk for future disease activity by establishing a reference database to derive reference values corrected for age and body-mass index (BMI). Furthermore, we used the reference database to test the suitability of sNfL as an endpoint for group-level comparison of effectiveness across disease-modifying therapies. METHODS For derivation of a reference database of sNfL values, a control group was created, comprising participants with no evidence of CNS disease taking part in four cohort studies in Europe and North America. We modelled the distribution of sNfL concentrations in function of physiological age-related increase and BMI-dependent modulation, to derive percentile and Z score values from this reference database, via a generalised additive model for location, scale, and shape. We tested the reference database in participants with multiple sclerosis in the Swiss Multiple Sclerosis Cohort (SMSC). We compared the association of sNfL Z scores with clinical and MRI characteristics recorded longitudinally to ascertain their respective disease prognostic capacity. We validated these findings in an independent sample of individuals with multiple sclerosis who were followed up in the Swedish Multiple Sclerosis registry. FINDINGS We obtained 10 133 blood samples from 5390 people (median samples per patient 1 [IQR 1-2] in the control group). In the control group, sNfL concentrations rose exponentially with age and at a steeper increased rate after approximately 50 years of age. We obtained 7769 samples from 1313 people (median samples per person 6��0 [IQR 3��0-8��0]). In people with multiple sclerosis from the SMSC, sNfL percentiles and Z scores indicated a gradually increased risk for future acute (eg, relapse and lesion formation) and chronic (disability worsening) disease activity. A sNfL Z score above 1��5 was associated with an increased risk of future clinical or MRI disease activity in all people with multiple sclerosis (odds ratio 3��15, 95% CI 2��35-4��23; p

This study analyzed the cerebrospinal fluid features of 31 coronavirus disease 2019 (COVID-19) patients with neurological complications. We observed neither severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in the cerebrospinal fluid, nor intrathecal immunoglobulin G (IgG) synthesis but did observe signs of blood-brain barrier disruption. These results might serve as a basis for a better understanding of SARS-CoV-2 related neuropathogenesis.

Additional file 1: Figure S1. MOG-induced EAE clinical score. (A) MOG35–55 induces EAE in B6 mice. EAE was induced by immunization with 200 μg of MOG35–55, emulsified in CFA on days 0. Mice also received 300 ng of pertussis toxin intravenously on days 0 and 2. EAE disease severity was followed, and incidence is shown in (B). (C) The forward scatter of both populations’ c-Met+ vs c-Met- CD8+ cells are represented. (D) Protein expression levels of c-Met by CD8+ T cells in EAE at day 14. Lines 1, 2, 3 correspond respectively to: 1) bulk splenic CD8+ T cells enriched by negative selection using μbeads, 2) FACS-sorted (EAE d14) c-Met+CD3+ CD8+ T cells and 3) FACS-sorted (EAE d14) c-Met-CD3+ CD8+ T cells, as measured by Western blot (n=3 mice). Purified CD8+ T cells were homogenized using a polytron in lysis buffer (50 mM Tris–HCl [pH 7.5], 250 mM NaCl, 1% Triton X‐100, 1 mM EDTA, and 1 mM DTT) containing complete protease inhibitors (Roche). Equal amounts (20 μg) of total protein from each sample were transferred to a 15% sodium dodecyl sulfate (SDS)–polyacrylamide gel and blotted onto anImmobilon‐P polyvinylidene difluoride (PVDF) membrane (Millipore). Expression levels of c‐Met were detected using properly diluted (1:100) mouse monoclonal anti‐c‐Met Ab (clone 3i20,Abcam), followed by a peroxidase‐conjugated secondary Ab to mouse IgG1 (eBioscience), and then visualized using chemiluminescence (Supersignal; Pierce). The blot was also probed with mouse monoclonal anti‐β‐actine (clone 15G5A11/E2) as a loading control (Sigma‐Aldrich).