Multi-parameter immune profiling of peripheral blood mononuclear cells by multiplexed single-cell mass cytometry in patients with early multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory demyelinating and neurodegenerative disease of the central nervous system (CNS). Studies in rodent models demonstrated an association of CNS-infiltrating monocyte-derived macrophages with disease severity. However, little is known about humans. Here, we performed an exploratory analysis of peripheral blood mononuclear cells (PBMCs) isolated from healthy controls and drug-naïve patients with early MS using multiplexed single-cell mass cytometry and algorithm-based data analysis. Two antibody panels comprising a total of 64 antibodies were designed to comprehensively analyse diverse immune cell populations, with particular emphasis on monocytes. PBMC composition and marker expression were overall similar between the groups. However, an increased abundance of CCR7+ and IL-6+ T cells was detected in early MS-PBMCs, whereas NFAT1hiT-bethiCD4+ T cells were decreased. Similarly, we detected changes in the subset composition of the CCR7+ and MIPßhi HLA-DR+ lymphocyte compartment. Only mild alterations were detected in monocytes/myeloid cells of patients with early MS, namely a decreased abundance of CD141hiIRF8hiCXCR3+CD68- dendritic cells. Unlike in Crohn's disease, no significant differences were found in the monocyte fraction of patients with early MS compared to healthy controls. This study provides a valuable resource for future studies designed to characterise and target diverse PBMC subsets in MS.

Uncovering convolutional neural network decisions for diagnosing multiple sclerosis on conventional MRI using layer-wise relevance propagation.

Machine learning-based imaging diagnostics has recently reached or even surpassed the level of clinical experts in several clinical domains. However, classification decisions of a trained machine learning system are typically non-transparent, a major hindrance for clinical integration, error tracking or knowledge discovery. In this study, we present a transparent deep learning framework relying on 3D convolutional neural networks (CNNs) and layer-wise relevance propagation (LRP) for diagnosing multiple sclerosis (MS), the most widespread autoimmune neuroinflammatory disease. MS is commonly diagnosed utilizing a combination of clinical presentation and conventional magnetic resonance imaging (MRI), specifically the occurrence and presentation of white matter lesions in T2-weighted images. We hypothesized that using LRP in a naive predictive model would enable us to uncover relevant image features that a trained CNN uses for decision-making. Since imaging markers in MS are well-established this would enable us to validate the respective CNN model. First, we pre-trained a CNN on MRI data from the Alzheimer's Disease Neuroimaging Initiative (n = 921), afterwards specializing the CNN to discriminate between MS patients (n = 76) and healthy controls (n = 71). Using LRP, we then produced a heatmap for each subject in the holdout set depicting the voxel-wise relevance for a particular classification decision. The resulting CNN model resulted in a balanced accuracy of 87.04% and an area under the curve of 96.08% in a receiver operating characteristic curve. The subsequent LRP visualization revealed that the CNN model focuses indeed on individual lesions, but also incorporates additional information such as lesion location, non-lesional white matter or gray matter areas such as the thalamus, which are established conventional and advanced MRI markers in MS. We conclude that LRP and the proposed framework have the capability to make diagnostic decisions of CNN models transparent, which could serve to justify classification decisions for clinical review, verify diagnosis-relevant features and potentially gather new disease knowledge.

Intrathecal IgM production is a strong risk factor for early conversion to multiple sclerosis.

OBJECTIVES: To evaluate intrathecal immunoglobulin M (IgM) production, as compared to previously established risk factors, as risk factor for conversion from clinically isolated syndrome (CIS) to multiple sclerosis (MS) and to explore the association of intrathecal IgM production with onset age and radiologic and CSF findings in CIS/early MS. METHODS: Comprehensive CSF data, including oligoclonal immunoglobulin G (IgG) bands (OCB) and calculated intrathecal IgM and IgG production, were collected in a prospective study of 150 patients with CIS/early MS with regular clinical and MRI assessments. RESULTS: Intrathecal IgM production >0% occurred in 23.2% (33/142) of patients, who were on average 5 years younger at disease onset (p = 0.013) and more frequently had infratentorial lesions (18/32, 56.3%) than patients without intrathecal IgM production (33/104, 31.7%, p = 0.021). In multivariable Cox regression analyses, intrathecal IgM production in patients with a CIS (n = 93, median clinical and MRI follow-up 24 and 21 months) was strongly associated with conversion to MS according to the McDonald 2010 criteria (hazard ratio [95% confidence interval] 3.05 [1.45-6.44], p = 0.003) after adjustment for age (0.96 [0.93-1.00], p = 0.059), OCB (0.92 [0.33-2.61], p = 0.879), intrathecal IgG production (0.98 [0.48-1.99], p = 0.947), and radiologic evidence of dissemination in space (2.63 [1.11-6.22], p = 0.028). CONCLUSION: Intrathecal IgM production is a strong independent risk factor for early conversion to MS and may thus represent a clinically meaningful marker for predicting future disease activity in patients with a CIS.

MRI Markers and Functional Performance in Patients With CIS and MS: A Cross-Sectional Study.

Introduction: Brain atrophy is a widely accepted marker of disease severity with association to clinical disability in multiple sclerosis (MS). It is unclear to which extent this association reflects common age effects on both atrophy and function. Objective: To explore how functional performance in gait, upper extremities and cognition is associated with brain atrophy in patients with Clinically Isolated Syndrome (CIS) and relapsing-remitting MS (RRMS), controlling for effects of age and sex. Methods: In 27 patients with CIS, 59 with RRMS (EDSS ≤3) and 63 healthy controls (HC), 3T MRI were analyzed for T2 lesion count (T2C), volume (T2V) and brain volumes [normalized brain volume (NBV), gray matter volume (NGMV), white matter volume (NWMV), thalamic volume (NThalV)]. Functional performance was measured with short maximum walking speed (SMSW speed), 9-hole peg test (9HPT) and symbol digit modalities test (SDMT). Linear regression models were created for functional variables with stepwise inclusion of age, sex and MR imaging markers. Results: CIS differed from HC only in T2C and T2V. RRMS differed from HC in NBV, NGMV and NThalV, T2C and T2V, but not in NWMV. A strong association with age was seen in HC, CIS and RRMS groups for NBV (r = -0.5 to -0.6) and NGMV (r = -0.6 to -0.8). Associations with age were seen in HC and RRMS but not CIS for NThalV (r = -0.3; r = -0.5), T2C (rs = 0.3; rs = 0.2) and T2V (rs = 0.3; rs = 0.3). No effect of age was seen on NWMV. Correlations of functional performance with age in RRMS were seen for SMSW speed, 9HPTand SDMT (r = -0.27 to -0.46). Regression analyses yielded significant models only in the RRMS group for 9HPT, SMSW speed and EDSS. These included NBV, NGMV, NThalV, NWMV, logT2V, age and sex as predictors. NThalV was the only MRI variable predicting a functional measure (9HPTr) with a higher standardized beta than age and sex (R2 = 0.36, p < 1e-04). Conclusion: Thalamic atrophy was a stronger predictor of hand function (9HPT) in RRMS, than age and sex. This underlines the clinical relevance of thalamic atrophy and the relevance of hand function as a clinical marker even in mildly disabled patients.

7 Tesla MRI of Balo's concentric sclerosis versus multiple sclerosis lesions.

BACKGROUND: Baló's concentric sclerosis (BCS) is a rare condition characterized by concentrically layered white matter lesions. While its pathogenesis is unknown, hypoxia-induced tissue injury and chemotactic stimuli have been proposed as potential causes of BCS lesion formation. BCS has been suggested to be a variant of multiple sclerosis (MS). Here, we aimed to elucidate similarities and differences between BCS and MS by describing lesion morphology and localization in high-resolution 7 Tesla (7 T) magnetic resonance imaging (MRI) scans. METHODS: Ten patients with Baló-type lesions underwent 7 T MRI, and 10 relapsing remitting MS patients served as controls. The 7 T MR imaging protocol included 3D T1-weighted (T1w) magnetization-prepared rapid gradient echo, 2D high spatial resolution T2*-weighted (T2*w) fast low-angle shot and susceptibility-weighted imaging. RESULTS: Intralesional veins were visible in the center of all but one Baló-type lesion. Four Baló-type lesions displayed inhomogeneous intralesional T2*w signal intensities, which are suggestive of microhemorrhages or small ectatic venules. Eight of 10 BCS patients presented with 97 additional lesions, 36 of which (37%) had a central vein. Lesions involving the cortical gray matter and the U-fibers were not detected in BCS patients. CONCLUSION: Our findings support the hypothesis that BCS and MS share common pathogenetic mechanisms but patients present with different lesion phenotypes.

Association of Retinal Ganglion Cell Layer Thickness With Future Disease Activity in Patients With Clinically Isolated Syndrome.

Importance: Clinically isolated syndrome (CIS) describes a first clinical incident suggestive of multiple sclerosis (MS). Identifying patients with CIS who have a high risk of future disease activity and subsequent MS diagnosis is crucial for patient monitoring and the initiation of disease-modifying therapy. Objective: To investigate the association of retinal optical coherence tomography (OCT) results with future disease activity in patients with CIS. Design, Setting, and Participants: This prospective, longitudinal cohort study took place between January 2011 and May 2017 at 2 German tertiary referral centers. A total of 179 patients with CIS were screened (80 in Berlin and 99 in Munich). Patients underwent neurological examination, magnetic resonance imaging (MRI), and OCT. Only eyes with no previous optic neuritis were considered for OCT analysis. Main Outcomes and Measures: The primary outcome was not meeting the no evidence of disease activity (NEDA-3) criteria; secondary outcomes were MS diagnosis (by the 2010 McDonald criteria) and worsening of disability. The primary measure was OCT-derived ganglion cell and inner plexiform layer thickness; the secondary measures included peripapillary retinal nerve fiber layer thickness, inner nuclear layer thickness, and MRI-derived T2-weighted lesions. Results: A total of 97 of the 179 screened patients (54.2%) were enrolled in the study at a median of 93 (interquartile range [IQR], 62-161) days after a first demyelinating event. The median follow-up duration (Kaplan-Meier survival time) was 729 (IQR, 664-903) days. Of 97 patients with CIS (mean age 33.6 [7.9] years; 61 [62.9%] female), 58 (59%) did not meet NEDA-3 criteria during the follow-up period. A Kaplan-Meier analysis showed a significant probability difference in not meeting NEDA-3 criteria by ganglion cell and inner plexiform later thickness (thinnest vs thickest tertile: hazard ratio [HR], 3.33 [95% CI, 1.70-6.55; P < .001; log-rank P = .001). A follow-up diagnosis of MS was more likely for patients with low ganglion cell and inner plexiform layer thickness (thinnest vs thickest tertile: HR, 4.05 [95% CI, 1.93-8.50]; P < .001). Low peripapillary retinal nerve fiber layer thickness likewise indicated risk of not meeting NEDA-3 criteria (thinnest vs thickest tertile: HR, 2.46 [95% CI, 1.29-4.66]; P = .01; log-rank P = .02). Inner nuclear layer thickness and T2-weighted lesion count were not associated with not meeting NEDA-3 criteria. Conclusions and Relevance: Retinal ganglion cell and inner plexiform layer thickness might prove a valuable imaging marker for anticipating future disease activity and diagnosis of MS in patients with CIS, which can potentially support patient monitoring and initiation of disease-modifying therapy.

Epstein-Barr virus antibodies in serum and DNA load in saliva are not associated with radiological or clinical disease activity in patients with early multiple sclerosis.

OBJECTIVE: To investigate the association of Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA-1) and viral capsid antigen (VCA) immunoglobulin (Ig)G antibodies in serum as well as EBV DNA load in saliva with radiological and clinical disease activity in patients with clinically isolated syndrome (CIS) and early relapsing-remitting MS (RRMS). METHODS: EBNA-1 and VCA immunoglobulin (Ig)G antibodies were determined in serum of 100 patients with CIS/early RRMS and 60 healthy controls. EBV DNA load was measured in saliva of 48 patients and 50 controls. Patients underwent clinical assessment with the Expanded Disability Status Scale (EDSS) and 3 Tesla magnetic resonance imaging at baseline and after a median of 20 months of follow-up (n = 63 for MRI, n = 71 for EDSS). The association of EBV parameters with occurrence of a second relapse, indicating conversion to clinically definite MS (CDMS), was evaluated over a median of 35 months of follow-up after the first clinical event (n = 89). RESULTS: EBNA-1 IgG antibody frequency (p = 0.00005) and EBNA-1 and VCA IgG antibody levels (p<0.0001 for both) were higher in patients than in controls. EBV DNA load in saliva did not differ between groups. Neither EBV antibody levels nor DNA load in saliva were associated with baseline or follow-up number or volume of T2-weighted (T2w) or contrast enhancing lesions, number of Barkhof criteria or the EDSS, or with the number of new T2w lesions, T2w lesion volume change or EDSS change on follow-up. Likewise, levels of EBV IgG antibodies in serum and DNA load in saliva were not associated with conversion to CDMS. CONCLUSIONS: While these findings confirm the association of EBV infection with early MS, neither EBNA-1 nor VCA IgG antibodies in serum nor EBV DNA load in saliva were associated with radiological or clinical disease activity in patients with CIS/early RRMS. These data are compatible with the concept that EBV may be a trigger for MS acting very early during the development of the disease.

Low 25-hydroxyvitamin D, but not the bioavailable fraction of 25-hydroxyvitamin D, is a risk factor for multiple sclerosis.

BACKGROUND AND PURPOSE: Low 25-hydroxyvitamin D [25(OH)D] levels correlate with higher disease activity in patients with multiple sclerosis (MS). However, it is not clear whether low 25(OH)D levels directly contribute to increased disease activity or merely represent a consequence of reduced endogenous vitamin D synthesis in more disabled MS patients. Furthermore, recent data suggest that bioavailable vitamin D, which also integrates the levels of vitamin D binding proteins and albumin, could be a biologically more relevant parameter than 25(OH)D. METHODS: Measured de-seasonalized 25(OH)D3 and vitamin D binding protein and calculated bioavailable and free vitamin D were compared in the baseline serum samples of 76 patients with clinically isolated syndrome enrolled in a longitudinal observational study and in 76 age- and sex-matched healthy controls (HC). RESULTS: 25(OH)D3 levels were lower in patients with clinically isolated syndrome (P = 0.002) than in HC, and more patients (8/76, 10.5%) than HC (1/76, 1.3%) had 25(OH)D3 levels <25 nmol/l (P = 0.03). In contrast, levels of 25(OH)D2, vitamin D binding protein and calculated levels of free and bioavailable vitamin D did not differ between the two groups. CONCLUSIONS: Lower 25(OH)D3 levels already in the earliest phase of disease and in clinically hardly affected patients suggest that low 25(OH)D3 levels are rather a risk factor for than a consequence of MS. Nevertheless, because bioavailable vitamin D levels did not differ between the two groups, the mechanism underlying the association of 25(OH)D3 and MS does not appear to be related to reduced bioavailability of vitamin D.

Next-generation sequencing identifies altered whole blood microRNAs in neuromyelitis optica spectrum disorder which may permit discrimination from multiple sclerosis.

BACKGROUND: Neuromyelitis optica spectrum disorder (NMOSD) and multiple sclerosis (MS) have a similar clinical phenotype but represent distinct diseases, requiring different therapies. MicroRNAs (miRNAs) are short non-coding RNAs whose expression profiles can serve as diagnostic biomarkers and which may be involved in the pathophysiology of neuroinflammatory diseases. Here, we analyzed miRNA profiles in serum and whole blood of patients with NMOSD and clinically isolated syndrome (CIS)/relapsing-remitting MS (RRMS) as well as healthy controls by next-generation sequencing (NGS). METHODS: MiRNA expression profiles were determined by NGS in sera of patients with aquaporin-4 antibody-positive NMOSD (n = 20), CIS/RRMS (n = 20), and healthy controls (n = 20) and in whole blood of patients with NMOSD (n = 11), CIS/RRMS (n = 60), and healthy controls (n = 43). Differentially expressed miRNAs were calculated by analysis of variance and t tests. All significance values were corrected for multiple testing. Selected miRNAs were validated in whole blood of patients with NMOSD (n = 18) and CIS/RRMS (n = 19) by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: None of 261 miRNAs detected in serum but 178 of 416 miRNAs detected in whole blood showed significantly different expression levels among the three groups. Pairwise comparisons revealed 115 (NMOSD vs. CIS/RRMS), 141 (NMOSD vs. healthy controls), and 44 (CIS/RRMS vs. healthy controls) miRNAs in whole blood with significantly different expression levels. qRT-PCR confirmed different expression levels in whole blood of patients with NMOSD and CIS/RRMS for 9 out of 10 exemplarily chosen miRNAs. In silico enrichment analysis demonstrated an accumulation of altered miRNAs in NMOSD in particular in CD15(+) cells (i.e., neutrophils and eosinophils). CONCLUSIONS: This study identifies a set of miRNAs in whole blood, which may have the potential to discriminate NMOSD from CIS/RRMS and healthy controls. In contrast, miRNA profiles in serum do not appear to be promising diagnostic biomarkers for NMOSD. Enrichment of altered miRNAs in CD15(+) neutrophils and eosinophils, which were previously implicated in the pathophysiology of NMOSD, suggests that miRNAs could be involved in the regulation of these cells in NMOSD.

Association of serum Epstein-Barr nuclear antigen-1 antibodies and intrathecal immunoglobulin synthesis in early multiple sclerosis.

Multiple sclerosis (MS) is associated with Epstein-Barr virus (EBV) infection. A characteristic feature of MS is an intrathecal synthesis of immunoglobulin (Ig)G. In 90 patients with clinically isolated syndromes/early relapsing-remitting MS, serum antibodies to Epstein-Barr nuclear antigen-1, but not to EBV viral capsid antigen, rubella, or varicella zoster virus, were higher (p=0.03) in those with than those without a calculated intrathecal IgG synthesis >0% and correlated with the percentage (r=0.27, p=0.009) and concentration (r=0.27, p=0.012) of intrathecally produced IgG. These findings suggest a link between EBV infection and the events leading to intrathecal IgG synthesis in patients with MS.