Prospective study validating a multidimensional treatment decision score predicting the 24-month outcome in untreated patients with clinically isolated syndrome and early relapsing-remitting multiple sclerosis, the ProVal-MS study.

INTRODUCTION: In Multiple Sclerosis (MS), patients´ characteristics and (bio)markers that reliably predict the individual disease prognosis at disease onset are lacking. Cohort studies allow a close follow-up of MS histories and a thorough phenotyping of patients. Therefore, a multicenter cohort study was initiated to implement a wide spectrum of data and (bio)markers in newly diagnosed patients. METHODS: ProVal-MS (Prospective study to validate a multidimensional decision score that predicts treatment outcome at 24 months in untreated patients with clinically isolated syndrome or early Relapsing-Remitting-MS) is a prospective cohort study in patients with clinically isolated syndrome (CIS) or Relapsing-Remitting (RR)-MS (McDonald 2017 criteria), diagnosed within the last two years, conducted at five academic centers in Southern Germany. The collection of clinical, laboratory, imaging, and paraclinical data as well as biosamples is harmonized across centers. The primary goal is to validate (discrimination and calibration) the previously published DIFUTURE MS-Treatment Decision score (MS-TDS). The score supports clinical decision-making regarding the options of early (within 6 months after study baseline) platform medication (Interferon beta, glatiramer acetate, dimethyl/diroximel fumarate, teriflunomide), or no immediate treatment (> 6 months after baseline) of patients with early RR-MS and CIS by predicting the probability of new or enlarging lesions in cerebral magnetic resonance images (MRIs) between 6 and 24 months. Further objectives are refining the MS-TDS score and providing data to identify new markers reflecting disease course and severity. The project also provides a technical evaluation of the ProVal-MS cohort within the IT-infrastructure of the DIFUTURE consortium (Data Integration for Future Medicine) and assesses the efficacy of the data sharing techniques developed. PERSPECTIVE: Clinical cohorts provide the infrastructure to discover and to validate relevant disease-specific findings. A successful validation of the MS-TDS will add a new clinical decision tool to the armamentarium of practicing MS neurologists from which newly diagnosed MS patients may take advantage. Trial registration ProVal-MS has been registered in the German Clinical Trials Register, `Deutsches Register Klinischer Studien` (DRKS)-ID: DRKS00014034, date of registration: 21 December 2018; https://drks.de/search/en/trial/DRKS00014034.

No evidence of oligoclonal bands, intrathecal immunoglobulin synthesis and B cell recruitment in acute ischemic stroke.

BACKGROUND: Within the past 10 years, immune mechanisms associated with acute ischemic stroke (AIS) have been brought into focus, but data on B cell activation and intrathecal Ig production is still scarce. In this study, we determined the prevalence of an elevated IgG index, positive oligoclonal bands (OCBs) and chemokine C-X-C motif ligand 13 (CXCL13) levels in the cerebrospinal fluid (CSF) as markers of intrathecal IgG synthesis and B cell activation in patients with AIS. METHODS: In a retrospective study we analyzed the cerebrospinal fluid (CSF) from 212 patients with AIS from December 2013 to May 2018 assessing intrathecal Ig synthesis, OCBs and CXCL13 concentrations. RESULTS: Overall, 5.7% (12/212) of AIS patients showed an intrathecal IgG synthesis, 0.5% (1/212) with isolated elevated IgG index, 5.2% (7/136) isolated positive OCBs and 2.9% (4/136) both elevated IgG index and positive OCBs. CXCL13 levels were elevated in 3.6% (3/83) of the patients. Approximately one third of these patients had simultaneously chronic inflammatory CNS disease (multiple sclerosis, neuromyelitis optica spectrum disorder, neurosarcoidosis). There was no significant association between CSF findings and stroke characteristics including vascular territory, localization, volume, etiology, acute treatment, or blood-brain barrier dysfunction. Intrathecal IgG synthesis was more common in patients with prior stroke. Longitudinal CSF analysis did not reveal any newly-occurring, but instead mostly persistent or even disappearing intrathecal IgG synthesis after AIS. CONCLUSIONS: We found no evidence of a relevant B cell recruitment and intrathecal IgG synthesis in patients with AIS. In fact, the occurrence of intrathecal IgG synthesis was associated with concurrent chronic inflammatory CNS disease or previous stroke. Consequently, in patients with first-ever AIS and intrathecal IgG synthesis, physicians should search for concomitant inflammatory CNS disease.

Cladribine treatment specifically affects peripheral blood memory B cell clones and clonal expansion in multiple sclerosis patients.

Introduction: B cells are acknowledged as crucial players in the pathogenesis of multiple sclerosis (MS). Several disease modifying drugs including cladribine have been shown to exert differential effects on peripheral blood B cell subsets. However, little is known regarding functional changes within the peripheral B cell populations. In this study, we obtained a detailed picture of B cell repertoire changes under cladribine treatment on a combined immunoglobulin (Ig) transcriptome and proteome level. Methods: We performed next-generation sequencing of Ig heavy chain (IGH) transcripts and Ig mass spectrometry in cladribine-treated patients with relapsing-remitting multiple sclerosis (n = 8) at baseline and after 6 and 12 months of treatment in order to generate Ig transcriptome and Ig peptide libraries. Ig peptides were overlapped with the corresponding IGH transcriptome in order to analyze B cell clones on a combined transcriptome and proteome level. Results: The analysis of peripheral blood B cell percentages pointed towards a significant decrease of memory B cells and an increase of naive B cells following cladribine therapy. While basic IGH repertoire parameters (e.g. variable heavy chain family usage and Ig subclasses) were only slightly affected by cladribine treatment, a significantly decreased number of clones and significantly lower diversity in the memory subset was noticeable at 6 months following treatment which was sustained at 12 months. When looking at B-cell clones comprising sequences from the different time-points, clones spanning between all three time-points were significantly more frequent than clones including sequences from two time-points. Furthermore, Ig proteome analyses showed that Ig transcriptome specific peptides could mostly be equally aligned to all three time-points pointing towards a proportion of B-cell clones that are maintained during treatment. Discussion: Our findings suggest that peripheral B cell related treatment effects of cladribine tablets might be exerted through a reduction of possibly disease relevant clones in the memory B cell subset without disrupting the overall clonal composition of B cells. Our results -at least partially- might explain the relatively mild side effects regarding infections and the sustained immune response after vaccinations during treatment. However, exact disease driving B cell subsets and their effects remain unknown and should be addressed in future studies.

Clinical-Radiological Mismatch in Multiple Sclerosis Patients during Acute Relapse: Discrepancy between Clinical Symptoms and Active, Topographically Fitting MRI Lesions.

BACKGROUND: Relapses in multiple sclerosis (MS) patients are usually defined as subacute clinical symptoms that last for at least 24 h. To validate a clinical relapse on magnetic resonance imaging (MRI), an anatomically fitting lesion with gadolinium enhancement in the central nervous system (CNS) would be mandatory. The aim of this study was to validate clinical relapses in regard to the concomitant detection of active, anatomically fitting MRI lesions. METHODS: We performed a retrospective analysis of 199 MS patients with acute relapse who had received an MRI scan before the initiation of methylprednisolone (MPS) therapy. Clinical data and MRIs were systematically reanalyzed by correlating clinical symptoms with their anatomical representation in the CNS. Patients were then categorized into subgroups with a clinical-radiological match (group 1) or clinical-radiological mismatch (group 2) between symptoms and active, topographically fitting lesions and further analyzed in regard to clinical characteristics. RESULTS: In 43% of our patients, we observed a clinical-radiological mismatch (group 2). Further analysis of patient characteristics showed that these patients were significantly older at the time of relapse. MS patients in group 2 also showed a significantly longer disease duration and significantly more previous relapses when compared to group 1. Comparing symptom clusters, the appearance of motor dysfunction during the current relapse was significantly more frequent in group 2 than in group 1. The overall dose of MPS treatment was significantly lower in group 2 than in group 1 with a similar treatment response in both groups. CONCLUSIONS: The substantial clinical-radiological mismatch during acute relapse in our study could be explained by several factors, including a psychosomatic component or disturbance of network connectivity. Alternatively, secondary progression or a diffuse neuro-inflammatory process might cause clinical symptoms, especially in older patients with a longer disease duration. As a consequence, treatment of clinical relapses and the definition of breakthrough disease should be reconsidered in regard to combined clinical and MRI criteria and/or additional biomarkers. Further studies are necessary to address the contribution of diffuse neuro-inflammation to the clinical presentation of symptoms.

Serum Neurofilament Light Chain and Glial Fibrillary Acidic Protein as Biomarkers in Primary Progressive Multiple Sclerosis and Hereditary Spastic Paraplegia Type 4.

In patients with slowly progressive spastic paraparesis, the differential diagnosis of primary progressive multiple sclerosis (PPMS) and hereditary spastic paraplegia (HSP) can be challenging. Serum neurofilament light chain (sNfL) and glial fibrillary acidic protein (sGFAP) are promising fluid biomarkers to support the diagnostic workup. Serum NfL is a marker of neuroaxonal decay sensitive to temporal changes, while elevated sGFAP levels may reflect astrocytal involvement in PPMS. We assessed sNfL and sGFAP levels in 25 patients with PPMS, 25 patients with SPG4 (the most common type of HSP) and 60 controls, using the highly sensitive single-molecule array (Simoa) platform. Patients were matched in age, sex, age at onset, disease duration and disease severity. Serum NfL levels were significantly increased in PPMS compared to SPG4 (p = 0.041, partial η² = 0.088), and there was a trend toward relatively higher sGFAP levels in PPMS (p = 0.097). However, due to overlapping biomarker values in both groups, we did not find sNfL and sGFAP to be useful as differential biomarkers in our cohort. The temporal dynamics indicate sNfL and sGFAP levels are most markedly elevated in PPMS in earlier disease stages, supporting their investigation in this group most in need of a diagnostic biomarker.

Next Generation Sequencing of Cerebrospinal Fluid B Cell Repertoires in Multiple Sclerosis and Other Neuro-Inflammatory Diseases-A Comprehensive Review.

During the last few decades, the role of B cells has been well established and redefined in neuro-inflammatory diseases, including multiple sclerosis and autoantibody-associated diseases. In particular, B cell maturation and trafficking across the blood-brain barrier (BBB) has recently been deciphered with the development of next-generation sequencing (NGS) approaches, which allow the assessment of representative cerebrospinal fluid (CSF) and peripheral blood B cell repertoires. In this review, we perform literature research focusing on NGS studies that allow further insights into B cell pathophysiology during neuro-inflammation. Besides the analysis of CSF B cells, the paralleled assessment of peripheral blood B cell repertoire provides deep insights into not only the CSF compartment, but also in B cell trafficking patterns across the BBB. In multiple sclerosis, CSF-specific B cell maturation, in combination with a bidirectional exchange of B cells across the BBB, is consistently detectable. These data suggest that B cells most likely encounter antigen(s) within the CSF and migrate across the BBB, with further maturation also taking place in the periphery. Autoantibody-mediated diseases, such as neuromyelitis optica spectrum disorder and LGI1 / NMDAR encephalitis, also show features of a CSF-specific B cell maturation and clonal connectivity with peripheral blood. In conclusion, these data suggest an intense exchange of B cells across the BBB, possibly feeding autoimmune circuits. Further developments in sequencing technologies will help to dissect the exact pathophysiologic mechanisms of B cells during neuro-inflammation.

Treatment of progressive multiple sclerosis with high-dose all-trans retinoic acid - no clear evidence of positive disease modifying effects.

BACKGROUND: All-trans retinoic acid (ATRA) is an acid derivative of vitamin A which is discussed as a promising candidate to ameliorate the disease course of multiple sclerosis (MS) by immunomodulation or even by promoting regeneration in progressive MS. Here we report a patient who significantly improved for MS related disability following administration of chemotherapy including ATRA for mitoxantrone-related acute promyelocytic leukemia and assess the effect of high-dose ATRA in three additional patients with progressive MS. METHODS: Patients with progressive MS who had failed previous therapies were treated with high-dose ATRA. Patients underwent clinical and routine laboratory monitoring. Additionally, PBMCs were analyzed by flow cytometry for lymphocyte subsets. RESULTS: ATRA was well tolerated and no pathological laboratory abnormalities were observed. After initial mild (not statistically significant) improvement of EDSS and mean MSFC z-score, ongoing disease progression was observed. One patient subacutely experienced severe cognitive and motor worsening. Cerebral MRI revealed persistent gadolinium-enhancing lesions. Flow cytometric alterations of peripheral blood naïve, central memory and effector memory CD4 and CD8 T cells, B lymphocytes, plasma cells, memory B cells, plasmablasts and natural killer (NK) cells did not reach statistical significance. CONCLUSIONS: Stand-alone therapy with ATRA did not ameliorate progressive MS in our limited cohort and we did not observe consistent alterations of T and B cell subsets. Intriguingly, application of ATRA may have caused marked disease exacerbation in one patient.

Specific Induction of Double Negative B Cells During Protective and Pathogenic Immune Responses.

Double negative (DN) (CD19+CD20lowCD27-IgD-) B cells are expanded in patients with autoimmune and infectious diseases; however their role in the humoral immune response remains unclear. Using systematic flow cytometric analyses of peripheral blood B cell subsets, we observed an inflated DN B cell population in patients with variety of active inflammatory conditions: myasthenia gravis, Guillain-Barré syndrome, neuromyelitis optica spectrum disorder, meningitis/encephalitis, and rheumatic disorders. Furthermore, we were able to induce DN B cells in healthy subjects following vaccination against influenza and tick borne encephalitis virus. Transcriptome analysis revealed a gene expression profile in DN B cells that clustered with naïve B cells, memory B cells, and plasmablasts. Immunoglobulin VH transcriptome sequencing and analysis of recombinant antibodies revealed clonal expansion of DN B cells that were targeted against the vaccine antigen. Our study suggests that DN B cells are expanded in multiple inflammatory neurologic diseases and represent an inducible B cell population that responds to antigenic stimulation, possibly through an extra-follicular maturation pathway.

Differentiation Between Guillain-Barré Syndrome and Acute-Onset Chronic Inflammatory Demyelinating Polyradiculoneuritis-a Prospective Follow-up Study Using Ultrasound and Neurophysiological Measurements.

Differentiation of Guillain-Barré syndrome (GBS) and acute-onset chronic inflammatory demyelinating polyradiculoneuritis (CIDP) might be intricate in early stages. We compared electrodiagnostics (EDx) and nerve ultrasound (NUS) as tools for early distinction and follow-up. NUS and EDx have been performed at first visitation and after 6 months. The nerve conduction study score (NCSS), the ultrasound pattern sum score (UPSS), and clinical scores were used for comparison. Compared with the 33 GBS patients, the 34 CIDP patients (50% with symptoms < 4 weeks) revealed significant nerve enlargement in ultrasound (p < 0.001) except for the roots and vagus, which exhibited increased values in both groups. EDx has no significant differences between both groups except for the A-wave frequency and the sural sparing pattern, which is more frequent in GBS (Fisher's exact p < 0.05). In the latter, particularly, pure sensory nerves were not enlarged in contrast to CIDP, in which those were mostly enlarged (p < 0.001). This ultrasonic sensory sparing pattern (uSSP) in combination with enlarged roots/vagus is the hallmark finding in GBS with sensitivity, specificity, and positive predictive value > 85%, whereas in CIDP, enlarged sensory und multifocally enlarged sensorimotor nerves are key differentiation features to GBS. Increased echointensity of the nerves further arises only in CIDP. After 6 months, in CIDP, the significant nerve enlargement persisted, whereas in GBS, all segments almost normalized. Clinical, ultrasonic, and NCS scores correlated significantly over the time. Enlarged roots/vagus in combination with uSSP might facilitate differentiation of GBS and CIDP in the early stage, and ultrasonic 6-month normalization underlines the diagnosis of GBS in cases of uncertainty. Trial Registration: DRKS-ID 00005253.

Validating Alzheimer's disease micro RNAs using next-generation sequencing.

INTRODUCTION: Molecular biomarkers for Alzheimer's disease (AD) can support detection and improved care for patients, but novel candidates require verification. We previously reported a 12-micro RNA (miRNA) blood-based signature using next-generation sequencing (NGS) of 54 AD cases and 22 controls. METHODS: We performed validation of 49 AD cases and 55 controls using NGS and also included 20 mild cognitive impairment and 90 multiple sclerosis samples to identify nonspecific markers. Thus, 103 AD cases, 77 unaffected controls, and 110 diseased controls were sequenced. Although the initial cohort came predominantly from the United States, the validation samples were collected in Germany. RESULTS: Five hundred eighty miRNAs were detected in the blood. In the initial cohort, we observed 203, in the validation cohort, 146 dysregulated miRNAs at a significance level of 0.05. With 68 miRNAs, the overlap was significant (P = .0003). Likewise, the area under the receiver operator characteristic curve values of the miRNAs correlated (correlation of 0.93; 95% confidence interval 0.89-0.96; P <10(-16)). DISCUSSION: MiRNAs have the potential to support AD diagnosis and patient care.

Low frequencies of central memory CD4 T cells in progressive multifocal leukoencephalopathy.

OBJECTIVES: To assess alterations in the composition of peripheral immune cells in acute progressive multifocal leukoencephalopathy (PML). METHODS: Fresh blood samples from 5 patients with acute PML and 10 healthy controls were analyzed by flow cytometry for naive, central memory and effector memory CD4 and CD8 T cells, B lymphocytes, plasma cells, memory B cells, plasma blasts, and natural killer (NK) cells. The frequency of central memory CD4 T cells was determined longitudinally during the course of PML in 2 patients. RESULTS: The frequencies of naive, central memory and effector memory CD8 T cells, B cells, plasma cells, and NK cells were not altered in patients with PML. In contrast, the frequencies of naive CD4 T cells (p = 0.04) and central memory CD4 T cells (p < 0.00001) were reduced and the frequencies of effector memory CD4 T cells were increased (p = 0.01). Longitudinal analysis showed that this pattern was preserved in a patient with fatal PML outcome and restored in one patient who recovered from PML. CONCLUSIONS: These data indicate that PML is associated with reduced frequencies of peripheral central memory helper T cells but not with alterations in the frequencies of cytotoxic T cell populations, B lymphocytes, plasma cells, or NK cells.

The intrinsic pathogenic role of autoantibodies to aquaporin 4 mediating spinal cord disease in a rat passive-transfer model.

Neuromyelitis optica (NMO) is causally linked to autoantibodies (ABs) against aquaporin 4 (AQP4). Here, we focused on the pathogenic effects exclusively mediated by human ABs to AQP4 in vivo. We performed cell-free intrathecal (i.th.) passive transfer experiments in Lewis rats using purified patient NMO immunoglobulin G (IgG) and various recombinant human anti-AQP4 IgG-ABs via implanted i.th. catheters. Repetitive application of patient NMO IgG fractions and of recombinant human anti-AQP4 ABs induced signs of spinal cord disease. Magnetic resonance imaging (MRI) revealed longitudinal spinal cord lesions at the site of application of anti-AQP4 IgG. Somatosensory evoked potential amplitudes were reduced in symptomatic animals corroborating the observed functional impairment. Spinal cord histology showed specific IgG deposition in the grey and white matter in the affected areas. We did not find inflammatory cell infiltration nor activation of complement in spinal cord areas of immunoglobulin deposition. Moreover, destructive lesions showing axon or myelin damage and loss of astrocytes and oligodendrocytes were all absent. Immunoreactivity to AQP4 and to the excitatory amino acid transporter 2 (EAAT2) was markedly reduced whereas immunoreactivity to the astrocytic marker glial fibrillary acid protein (GFAP) was preserved. The expression of the NMDA-receptor NR1 subunit was downregulated in areas of IgG deposition possibly induced by sustained glutamatergic overexcitation. Disease signs and histopathology were reversible within weeks after stopping injections. We conclude that in vivo application of ABs directed at AQP 4 can induce a reversible spinal cord disease in recipient rats by inducing distinct histopathological abnormalities. These findings may be the experimental correlate of "penumbra-like" lesions recently reported in NMO patients adjacent to effector-mediated tissue damage.