Serum neurofilament light chain (sNfL) and glial fibrillary acidic protein (sGFAP) are promising multiple sclerosis (MS) biomarkers, but biological confounding and inter-assay variability limit real-world use. We assessed whether Z score normalisation enables platform-agnostic monitoring in people with MS (pwMS) under ocrelizumab, and which early timepoint best stratifies subsequent biomarker trajectories. This pooled multicentre analysis of three German cohorts included pwMS treated with ocrelizumab for ≥ 12 months. Elecsys® or Simoa® assays were harmonised using covariate-adjusted Z scores. We evaluated biomarker 24-month trajectories and whether stratification and relative reductions predicted 24-month biomarker-status. 430 pwMS were included [78

BACKGROUND AND OBJECTIVES:Disability trajectories in aquaporin-4 immunoglobulin G-seropositive neuromyelitis optica spectrum disorder (AQP4-IgG+ NMOSD) and myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) are primarily driven by attack-related damage. Confirmed disability worsening (CDW) independent of attacks has been described but occurs infrequently in AQP4-IgG+ NMOSD and MOGAD. Confirmed disability improvement (CDI) has not been evaluated in large cohorts. We determined the frequency of CDI and CDW independent of attacks and identified clinical factors associated with these outcomes in AQP4-IgG+ NMOSD and MOGAD. METHODS:This retrospective, multicenter cohort study analyzed data from the German Neuromyelitis Optica Study Group (NEMOS) registry. Adult patients with AQP4-IgG+ NMOSD or MOGAD and longitudinal Expanded Disability Status Scale (EDSS) assessments were included. EDSS episodes were defined as periods with ≥3 EDSS assessments without attacks, obtained ≥90 days after attack. CDW and CDI were defined as sustained EDSS increase or decrease (≥1.5 for baseline EDSS 0; ≥1.0 for EDSS 1.0-5.5; ≥0.5 for EDSS ≥6.0) confirmed after at least 6 months. The primary outcomes were annualized CDI and CDW rates. Risk factors were assessed using multivariable Anderson-Gill regression models. RESULTS:A total of 338 EDSS episodes of 307 patients (n: 202/105, median age at EDSS change: 56/41 years, 88/49% female, both p < 0.001; AQP4-IgG+ NMOSD/MOGAD) were included. Adjusted annualized CDI and CDW rates did not differ between AQP4-IgG+ NMOSD (CDI: 0.083, 95% CI 0.029-0.233; CDW: 0.025, 95% CI 0.007-0.092) and MOGAD (CDI: 0.057, 95% CI 0.012-0.277; CDW: 0.036, 95% CI 0.002-0.513). In AQP4-IgG+ NMOSD, a lower number of prior attacks was associated with higher CDI rates (hazard ratio [HR] 0.89, 95% CI 0.82-0.97). Younger age was associated with increased CDI rates in both AQP4-IgG+ NMOSD and MOGAD (HR 0.96, 95% CI 0.94-0.99, for both). DISCUSSION:CDI and CDW independent of attacks, although rare, occur in AQP4-IgG+ NMOSD and MOGAD. The association between fewer prior attacks and higher CDI rates in AQP4-IgG+ NMOSD underscores the importance of early attack prevention. Limitations include the retrospective design, and the limited number of CDI and CDW events.

Introduction Advances in ultrasensitive assay techniques have enabled precise quantification of serum neurofilament light chain (sNfL) and serum glial fibrillary acidic protein (sGFAP), highlighting their potential as dynamic biomarkers for detecting neuroaxonal injury, disease activity and progression in multiple sclerosis (MS). In the NeuroFilMS study, sNfL is being investigated prospectively as a prognostic biomarker for clinical and radiological disease activity in relapsing MS, while sGFAP is retrospectively explored as a marker of disease progression. The aim is to assess whether longitudinal monitoring of sNfL can inform diagnostic and therapeutic decisions in the treatment of people with MS (pwMS) and whether retrospective sGFAP measurements provide additional insights into disease progression. The study additionally aims to evaluate the comparability of different assay methods.Methods and analysis NeuroFilMS is a prospective, multicentre study that will be conducted across multiple MS study centres throughout Germany, in collaboration with the German Multiple Sclerosis Registry set up by the German National Multiple Sclerosis Society (Deutsche Multiple Sklerose Gesellschaft). The study aims to enrol 1500 pwMS diagnosed with relapsing MS. Participants will be randomised in a 2:1 ratio (n=1000 vs n=500) to either immediate sNfL reporting or delayed sNfL reporting to treating physicians, in order to evaluate how sNfL availability influences therapeutic decision-making in routine healthcare regarding diagnostics and therapy decisions. Over a 2-year follow-up period, pwMS will attend three study visits integrated into routine care, including blood sampling for sNfL, clinical evaluations and routine MRI assessments; sGFAP will be measured retrospectively in batches, as it is not currently available for routine diagnostic use. The study follows the standardised protocols for biosample collection, performed both within clinical routine laboratory procedures and through research collaboration. Statistical analyses will involve both descriptive and inferential methods to evaluate biomarker performance and clinical associations.Ethics and dissemination The study has received ethical approval from the Clinical Ethics Committee of Charité–Universitätsmedizin Berlin (EA4/136/24) and will be conducted in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines. Findings will be disseminated through peer-reviewed publications, conference presentations and engagement with patient organisations and clinical networks.Trial registration number DRKS00034337.

INTRODUCTION:Rituximab is effective and widely used as long-term treatment in aquaporin-4-IgG-positive neuromyelitis optica spectrum disorder (AQP4-IgG+ NMOSD). However, infections remain a significant concern during rituximab treatment. METHODS:We conducted a retrospective multicenter cohort study within the NMO Study Group (NEMOS) in Germany, analyzing demographic and clinical data from people with AQP4-IgG+ NMOSD receiving rituximab or azathioprine by retrospective chart, and compared infection occurrence and severity. For rituximab-treated patients, we collected laboratory data (blood lymphocytes, B-cell counts, serum IgG, IgM, and IgA levels), assessed risk factors for infections, and determined the probability of infection within a 3-month window before and after the laboratory assessment. RESULTS:In 92/170 rituximab and in 12/33 azathioprine treatment episodes, one or more infections were documented. Rituximab and azathioprine showed comparable types and risk of infection (HR = 1.24, 95% CI: 0.68-2.25). Rituximab-treated individuals older than 60 years had a higher risk of infection (HR = 1.62, 95% CI: 1.02-2.57). Hypogammaglobulinemia (IgG < 6.0 g/L: OR = 2.27, 95% CI: 1.15-4.48; IgM < 0.3 g/L: OR = 2.08, 95% CI: 1.05-4.09) predicted infections and the occurrence of both low IgG and IgM serum levels further increased the risk of infection (OR = 2.77, 95% CI: 1.10-6.98) during rituximab treatment. Low IgG and IgA serum levels as well as lymphopenia predicted infection-related hospitalizations. CONCLUSION:Age > 60 years and immunoglobulin serum levels during rituximab treatment may serve as predictors for infection and help to individualize treatment decisions in NMOSD.

When immune cells interact, they frequently exchange membrane-bound antigens. Our evolving understanding of these processes challenges the cellular specificity of lineage markers and therapeutic monoclonal antibodies. By using mouse and human B-T cell co-cultures, we report that CD19, an assumingly exclusive B cell marker, is transferred via trogocytosis when B cells activate T cells. In a B cell-driven model of experimental autoimmune encephalomyelitis, CD19+ T cells expand and show enhanced features of activation, differentiation, and encephalitogenic potential ex vivo. Additionally, co-transfer of CD19 and functional IgM from B cells results in the gain of B cell function by T cells. In patients with chronic central nervous system (CNS) demyelination, CD19+ T cells display a pro-inflammatory phenotype and are concomitantly depleted by inebilizumab, an approved anti-CD19 antibody, which raises important considerations for the therapeutic use of monoclonal antibodies overall. Finally, we report that myeloid cells acquire CD19 and functional IgM after phagocytosis of apoptotic B cells and thereby gain functional B cell properties. These findings highlight the commonness of membrane and antigen-transfer between cells, resulting in transmission of cellular function.

BACKGROUND AND OBJECTIVES:Neuromyelitis optica spectrum disorders (NMOSDs) comprise inflammatory processes of the CNS. Most patients with NMOSD have serum immunoglobulin (Ig) G autoantibodies directed against the astrocytic water channel aquaporin-4 (AQP4-IgG). In this study, we analyzed HLA allelic frequencies in a large cohort of patients with NMOSD, stratified by ethnicity and AQP4-IgG status, compared with healthy controls. METHODS:Next-generation sequencing-based HLA class I and II genotyping was performed in 174 White, 45 Black, and 41 Hispanic AQP4-IgG-positive (AQP4-IgG+) patients with NMOSD; 49 White patients with AQP4-IgG-negative (AQP4-IgG-) NMOSD; and 2,427 White, 244 Black, and 155 Hispanic controls. Correction for multiple testing was performed using the Bonferroni method. RESULTS:In White AQP4-IgG+ patients with NMOSD, the most significantly associated alleles were HLA-DQA1*05:01:01 (30.1% vs 11.1%, odds ratio 3.43 [95% CI 2.65-4.42], corrected p = 8.95E-17), HLA-DQB1*02:01:01 (29.9% vs 11.3%, 3.34 [2.58-4.31], corrected p = 4.33E-16), and HLA-DRB1*03:01:01 (29.2% vs 11.6%, 3.15 [2.43-4.06], corrected p = 3.66E-14), followed by HLA-B*08:01:01 (26% vs 10.4%, 3.02 [2.3-3.94] corrected p = 8.79E-12), HLA-C*07:01:01 (27.8% vs 14%, 2.36 [1.81-3.04], corrected p = 2.44E-07), and HLA-DRB3*01:01:02 (28% vs 14.7%, 2.27 [1.73-2.95], corrected p = 2.19E-06). The frequency of HLA-DRB1*08:04:01 was higher in Black AQP4-IgG+ patients with NMOSD than in Black controls but did not achieve statistical significance (19.3% vs 5.7%, 3.92 [1.91-7.86], corrected p = 0.08). Nevertheless, when compared with a larger cohort of Black controls (n = 16,178), the frequency of HLA-DRB1*08:04 (19.3% vs 5.1%, 4.46 [2.45-7.66], corrected p = 1.88E-03) was significantly higher in Black AQP4-IgG+ patients with NMOSD. No significant HLA associations were detected in AQP4-IgG+ Hispanic patients or White AQP4-IgG- patients with NMOSD. DISCUSSION:This study confirms the previously recognized association of HLA-DRB1*03:01 with AQP4-IgG+ NMOSD in White patients and extends this association to the HLA-DRB1*03:01:01∼HLA-DQA1*05:01:01∼HLA-DQB1*02:01:01 haplotype. Furthermore, it identifies an association of HLA-DRB1*08:04 with AQP4-IgG+ NMOSD in Black patients. However, no HLA associations were detected in White AQP4-IgG- patients with NMOSD. The immunogenetic differences between AQP4-IgG+ and AQP4-IgG- NMOSD support pathophysiologic distinctions between these entities.

Background:Anti-CD20 therapies are widely used in patients with multiple sclerosis (pwMS). Recognition of rare adverse reactions to these therapies is therefore important. Objectives:To report dermatological and gastrointestinal adverse reactions in a single-center cohort of ocrelizumab treated pwMS. Methods:Retrospective analysis conducted at a multiple sclerosis outpatient clinic of Charité - Universitätsmedizin Berlin, Berlin, Germany, between March 2020 and February 2025. Results:Among 447 ocrelizumab treated pwMS, 8 (1.8%) developed dermatological adverse reactions after a median (range) of 20.5 (6-72) months following start of therapy, including lichen planus (n=2), rosacea (n=1), psoriatic arthritis (n=1), guttate psoriasis (n=1), psoriasis vulgaris (n=1), nail psoriasis (n=1) and palmoplantar psoriasis (n=1). Another 5 (1.1%) patients developed gastrointestinal adverse reactions 24 (0.25-77) months after starting therapy, including Crohn's disease (n=1), toxic colitis (n=1), lymphocytic colitis (n=1), perforated appendicitis (n=1) and acute cholecystitis (n=1). Due to these adverse reactions, ocrelizumab was stopped in 7/13 patients. At last follow-up, adverse reactions had completely improved in 4/13, incompletely improved in 6/13, and persisted in 3/13 patients. Conclusions:Clinicians should be aware of dermatological and gastrointestinal adverse reactions associated with ocrelizumab, which can develop from a few weeks up to six years after start of therapy.

Abstract Recurrent inflammatory attacks in AQP4-antibody-seropositive neuromyelitis optica spectrum disorder (AQP4-IgG+NMOSD) can lead to devastating disabilities such as visual and motor dysfunction, pain, and cognitive impairment. However, the mechanisms driving the long-term effects of attacks and potential for subsequent recovery are still not well understood after patients enter a relapse-free disease phase. Here, we leveraged advanced structural and diffusion-weighted imaging analyses in a longitudinal cohort of patients with “stable” AQP4-IgG+NMOSD (retrospectively assessed ≥ 12 months without attack, n =33, 31/33 female, mean age 49.7 years (SD 14.2)). Brain changes over a median of 4 annual visits (range 2-6) were evaluated using FreeSurfer-based volumetry, regional damage profiles of white matter fibre bundles, cognitive testing (BRB-N), and neuropsychiatric self-reports. Our analysis revealed four key findings: (1) In the absence of new attacks, pre-existing symptoms persisted and contributed to motor impairment, fatigue, and lower visual function. By contrast, cognitive impairment - selective to higher attention and processing speed - improved over time (PASAT3s, P FDR =0.007). (2) On a macroscopic brain level, the continued decline of whole brain volumes ( P FDR =0.037) was mainly driven by loss of cortical grey matter ( P FDR =0.013) and linked to poorer motor outcomes (9-hole peg test: ρₛ =-0.55, P FDR =0.021) and higher pain levels (PD-Q: ρₛ =-0.51, P FDR =0.021) at last follow-up. Large-scale age- and sex-stratified reference curves ( Braincharts ) confirmed that cortical atrophy exceeded normal ageing. (3) Thalamic volumes, by contrast, were significantly higher compared to those of healthy participants ( P FDR =0.044) throughout the entire follow-up period and predicted more favourable long-term attention (SDMT: ρₛ =0.63, P FDR =0.003) and spatial memory outcomes (SPART sum score: ρₛ =0.62, P FDR =0.029) as early as at the first MRI. Larger thalamic volumes were mainly seen in a subgroup of younger patients with lower disability burden, fewer comorbidities, and better integrity of thalamus-adjacent white matter tracts. (4) On a microstructural level, tract-specific longitudinal patterns emerged: decreasing regional fractional anisotropy (FA) in the optic radiation, thalamo-prefrontal and thalamo-occipital projections was linked to worse cognitive outcomes (e.g., SDMT: ρₛ =0.62, P FDR =0.012), while increasing FA, particularly in the corticospinal tract and inferior fronto-occipital fasciculus, predicted more favourable long-term cognitive and visual functions (e.g., NEI VFQ-25: ρₛ =0.64, P FDR =0.011). Collectively, our data suggest that even in relapse-free AQP4-IgG+NMOSD there is evidence for declining cortical volume, thalamic reserve in some patients, and white matter microstructural damage in distinct regions. Our clinically relevant findings elucidated in the “stable” disease phase highlight longitudinal mechanisms contributing to the long-term prognoses of patients with AQP4-IgG+NMOSD.

Abstract Intrathecal antibody synthesis is a hallmark of multiple sclerosis (MS). Although some intrathecally synthesized antibodies in MS are known to target viral antigens, the spectrum of their antigenic specificities remains incompletely defined. We combined proteome-wide antibody profiling by Phage ImmunoPrecipitation Sequencing (PhIP-Seq) with cross-compartment analytics (MICAR) to study intrathecal antibody synthesis at peptide resolution in paired CSF and serum samples from individuals with MS ( n = 40) and non-MS controls ( n = 83). While intrathecal antibody responses in MS were polyspecific and included reactivities to various viruses, we identified a subset of individuals with a convergent intrathecal antibody reactivity to a previously described motif within the Epstein-Barr virus (EBV) protein BRRF2 (BRRF2 408–415 ). This motif-directed response showed co-reactivity with multiple CNS-expressed human antigens. In an independent cohort of n = 909 individuals with MS and n = 311 controls, including individuals with NMOSD and MOGAD, serum antibodies to BRRF2 408–415 were detected in 8.36% of MS individuals and in 0.64% of non-MS controls, corresponding to an odds ratio for MS of 14.1 (95% CI: 4.4–86.04). Within MS individuals, BRRF2 408–415 seropositivity was associated with increased intrathecal IgG synthesis. Cross-reactivity of antibodies to BRRF2 408–415 with human targets, including TRIM71 and RTN2, was confirmed by competition ELISA and cell-based assays. Together, these data define an intrathecal EBV BRRF2-linked antibody signature with human target cross-reactivity in a subset of MS individuals. This signature identifies a highly specific serological marker in MS and may support future stratification of the heterogeneous MS spectrum.

OBJECTIVE:To evaluate the diagnostic accuracy of glial fibrillary acidic protein (GFAP) measured in dried plasma spots versus conventional plasma- and serum-GFAP testing for assessment of disease severity in aquaporin-4 immunoglobulin G-positive neuromyelitis optica spectrum disorder (AQP4-IgG+ NMOSD). METHODS:A neuroimmunological prospective cohort of remission samples from 70 participants with the diagnoses AQP4-IgG+ NMOSD (n = 19), myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD; n = 9), relapsing-remitting multiple sclerosis (RRMS; n = 28) and healthy controls (HC; n = 14) from a single center were included. GFAP concentrations were measured in frozen and thawed plasma and paired dried plasma spot (DPS) samples using an ultrasensitive proximity ligation-based assay (nucleic acid-linked immuno-sandwich assay; NULISA). In NMOSD, GFAP was additionally quantified in paired serum samples using a single-molecule array (Simoa) assay as a reference. Cross-matrix correlations, diagnostic performance, and associations with neurological disability were evaluated. RESULTS:GFAP concentrations measured by NULISA correlated strongly between plasma and DPS samples across diagnostic groups and healthy controls. In NMOSD, plasma GFAP measured by NULISA showed strong concordance with serum GFAP quantified by Simoa. DPS-derived and plasma-derived GFAP demonstrated good diagnostic accuracy for AQP4-IgG+ NMOSD and was significantly associated with neurological disability as measured by the Expanded Disability Status Scale (EDSS). Group-wise comparisons across plasma and DPS showed retained elevation of GFAP in AQP4-IgG+ NMOSD compared with the other diagnostic groups and healthy controls. INTERPRETATION:GFAP quantification using the NULISA platform is feasible in plasma and DPS samples and enables reliable biomarker assessment in a potentially remote-compatible setting. DPS-derived GFAP measurements retained meaningful information on disability in NMOSD and may provide an analytical framework for future studies evaluating minimally invasive capillary or self-sampling approaches.

BACKGROUND:Data on cognition in adult patients with myelin oligodendrocyte glycoprotein antibody-associated disease (pwMOGAD) are scarce. OBJECTIVE:To examine cognitive function in pwMOGAD and assess relative risks (RR) for cognitive impairment (CImp) in pwMOGAD relative to healthy controls (HC), aquaporin 4-immunoglobulin G positive neuromyelitis optica spectrum disorders (pwAQP4+NMOSD), and double-seronegative NMOSD (pwdsNMOSD) compared to HC. METHODS:Data derived from a cohort with neuroimmunological disorders. Cognitive performance was assessed using Rao's brief repeatable battery of neuropsychological tests, compared to HC using confounder-adjusted linear regressions. CImp was defined as performing two standard deviations below the HC mean in any subtest. RR for CImp was calculated using generalized linear models. RESULTS:We evaluated cognitive performance of 21 pwMOGAD and 25 HC. CImp was additionally determined in 43 pwAQP4+NMOSD and 15 pwdsNMOSD. PwMOGAD performed worse on Selective Reminding Test, and the symbol digit modalities test compared to HC. Adjusted RR for CImp were 1.9 (95% CI 0.9-4.1) in pwMOGAD, 1.9 (95% CI 1.0-3.9) in pwAQP4+NMOSD and 2.1 (95% CI 0.9-4.6) in pwdsNMOSD. CONCLUSION:pwMOGAD performed worse in information processing speed, verbal learning, storage and retrieval compared to HC. RR for CImp in pwMOGAD compared to HC was similar to that estimated for pwAQP4+NMOSD and pwdsNMOSD.

BACKGROUND:We compared the prognostic value of serum neurofilament light chain (sNFL) and glial fibrillary acidic protein (sGFAP) for clinical and radiologic disease activity among patients with clinically isolated syndrome or early multiple sclerosis (MS) with optic neuritis (pwON) and non-optic neuritis (pwNON) as the first manifestation. METHODS:Patients within 7 months (pwON and pwNON) from disease onset and patients with MS with a disease duration longer than 12 months (pwMS) as controls were included. sNFL and sGFAP were analyzed at baseline and at follow-ups using SIMOA technology. Linear mixed models and Cox regression analyses were applied. RESULTS:We included 165 samples of 86 patients (18 pwON, 46 pwNON, 21 pwMS). Median follow-up time was 80 months. Mean sNFL z scores were higher in pwNON (1.06) than pwON (0.53) and pwMS (0.94). In pwNON, but not pwON, higher sNFL z scores were associated with an elevated risk for a subsequent attack (pwNON: HR 1.63 [95% CI 1.12 to 2.27], p = 0.005; pwON: HR 0.80 [95% CI 0.51-2.27], p = 0.318) and new MRI T2 lesions (pwNON: HR 1.66 [95% CI 1.31 to 2.11], p < 0.001; pwON: HR 1.16 [95% CI 0.82 to 1.63], p = 0.404). sGFAP z scores were associated with a lower risk of a subsequent attack in pwON (HR 0.34 [95% CI 0.12 to 0.98], p = 0.047). CONCLUSION:sNFL but not sGFAP predicted future clinical and MRI disease activity only in pwNON, potentially suggesting that the prognostic value of sNFL may depend on the type of the first manifestation.

Background: We conducted a retrospective in silico analysis of routine laboratory data (RISAROLDA) to study the association of Epstein-Barr virus (EBV) and multiple sclerosis (MS). Methods: Patients with MS and 10 different inflammatory/neoplastic diseases were identified by ICD10 codes. Results of routine laboratory testing for antibodies to EBV, measles, mumps, rubella, herpes simplex virus, varicella zoster virus and cytomegalovirus were extracted using a digital tool. Results: Among 10,669 patients with MS and 42,222 controls, EBV serologies were available from 492 (4.6%) patients with MS and 1918 (4.5%) controls. While all but three patients with an ICD10 diagnosis of MS were EBV seropositive, closer inspection of the three EBV seronegative patients revealed they were misdiagnosed with MS, resulting in a 100% EBV seroprevalence in the remaining 489 patients with MS. In contrast, EBV seroprevalences were lower in all other diseases (78.6%-97.8%). Serum antibodies to the Epstein-Barr nuclear antigen-1, but not to the viral capsid antigen, were higher in patients with MS than in all other diseases. In patients with MS, seroprevalences of all other common viruses were lower than those of EBV, but the frequency of intrathecal production of antibodies to EBV was lower than that of other common viruses. Conclusions: These findings suggest that the association of EBV and MS is specific for MS as compared to various other inflammatory/neoplastic diseases and that a negative EBV serology might be a marker for the absence of MS. RISAROLDA is a powerful approach for the screening of real-world laboratory data.