Cerebrospinal fluid findings in patients with myelin oligodendrocyte glycoprotein (MOG) antibodies. Part 1: Results from 163 lumbar punctures in 100 adult patients.

BACKGROUND: New-generation cell-based assays have demonstrated a robust association of serum autoantibodies to full-length human myelin oligodendrocyte glycoprotein (MOG-IgG) with (mostly recurrent) optic neuritis, myelitis, and brainstem encephalitis, as well as with neuromyelitis optica (NMO)-like or acute-disseminated encephalomyelitis (ADEM)-like presentations. However, only limited data are yet available on cerebrospinal fluid (CSF) findings in MOG-IgG-associated encephalomyelitis (MOG-EM; also termed MOG antibody-associated disease, MOGAD). OBJECTIVE: To describe systematically the CSF profile in MOG-EM. MATERIAL AND METHODS: Cytological and biochemical findings (including white cell counts and differentiation; frequency and patterns of oligoclonal bands; IgG/IgM/IgA and albumin concentrations and CSF/serum ratios; intrathecal IgG/IgA/IgM fractions; locally produced IgG/IgM/IgA concentrations; immunoglobulin class patterns; IgG/IgA/IgM reibergrams; Link index; measles/rubella/zoster (MRZ) reaction; other anti-viral and anti-bacterial antibody indices; CSF total protein; CSF L-lactate) from 163 lumbar punctures in 100 adult patients of mainly Caucasian descent with MOG-EM were analyzed retrospectively. RESULTS: Most strikingly, CSF-restricted oligoclonal IgG bands, a hallmark of multiple sclerosis (MS), were absent in almost 90% of samples (N = 151), and the MRZ reaction, the most specific laboratory marker of MS known so far, in 100% (N = 62). If present, intrathecal IgG (and, more rarely, IgM) synthesis was low, often transient and mostly restricted to acute attacks. CSF WCC was elevated in > 50% of samples (median 31 cells/µl; mostly lymphocytes and monocytes; > 100/µl in 12%). Neutrophils were present in > 40% of samples; activated lymphocytes were found less frequently and eosinophils and/or plasma cells only very rarely (< 4%). Blood-CSF barrier dysfunction (as indicated by an elevated albumin CSF/serum ratio) was present in 48% of all samples and at least once in 55% of all patients (N = 88) tested. The frequency and degree of CSF alterations were significantly higher in patients with acute myelitis than in patients with acute ON and varied strongly depending on attack severity. CSF L-lactate levels correlated significantly with the spinal cord lesion load in patients with acute myelitis (p < 0.0001). Like pleocytosis, blood-CSF barrier dysfunction was present also during remission in a substantial number of patients. CONCLUSION: MOG-IgG-positive EM is characterized by CSF features that are distinct from those in MS. Our findings are important for the differential diagnosis of MS and MOG-EM and add to the understanding of the immunopathogenesis of this newly described autoimmune disease.

Human Brain Endothelial CXCR2 is Inflammation-Inducible and Mediates CXCL5- and CXCL8-Triggered Paraendothelial Barrier Breakdown.

Chemokines (C-X-C) motif ligand (CXCL) 5 and 8 are overexpressed in patients with multiple sclerosis, where CXCL5 serum levels were shown to correlate with blood-brain barrier dysfunction as evidenced by gadolinium-enhanced magnetic resonance imaging. Here, we studied the potential role of CXCL5/CXCL8 receptor 2 (CXCR2) as a regulator of paraendothelial brain barrier function, using the well-characterized human cerebral microvascular endothelial cell line hCMEC/D3. Low basal CXCR2 mRNA and protein expression levels in hCMEC/D3 were found to strongly increase under inflammatory conditions. Correspondingly, immunohistochemistry of brain biopsies from two patients with active multiple sclerosis revealed upregulation of endothelial CXCR2 compared to healthy control tissue. Recombinant CXCL5 or CXCL8 rapidly and transiently activated Akt/protein kinase B in hCMEC/D3. This was followed by a redistribution of tight junction-associated protein zonula occludens-1 (ZO-1) and by the formation of actin stress fibers. Functionally, these morphological changes corresponded to a decrease of paracellular barrier function, as measured by a real-time electrical impedance-sensing system. Importantly, preincubation with the selective CXCR2 antagonist SB332235 partially prevented chemokine-induced disturbance of both tight junction morphology and function. We conclude that human brain endothelial CXCR2 may contribute to blood-brain barrier disturbance under inflammatory conditions with increased CXCL5 and CXCL8 expression, where CXCR2 may also represent a novel pharmacological target for blood-brain barrier stabilization.

Pathogenic inflammation in the CNS of mice carrying human PLP1 mutations.

Progressive forms of multiple sclerosis lead to chronic disability, substantial decline in quality of life and reduced longevity. It is often suggested that they occur independently of inflammation. Here we investigated the disease progression in mouse models carrying PLP1 point mutations previously found in patients displaying clinical features of multiple sclerosis. These mouse models show loss-of-function of PLP1 associated with neuroinflammation; the latter leading to clinically relevant axonal degeneration, neuronal loss and brain atrophy as demonstrated by inactivation of the recombination activating gene 1. Moreover, these pathological hallmarks were substantially amplified when we attenuated immune regulation by inactivation of the programmed cell death-1 gene. Our observations support the view that primary oligodendroglial abnormalities can evoke pathogenically relevant neuroinflammation that drives neurodegeneration, as observed in some forms of multiple sclerosis but also in other, genetically-mediated neurodegenerative disorders of the human nervous system. As many potent immunomodulatory drugs have emerged during the last years, it is tempting to consider immunomodulation as a treatment option not only for multiple sclerosis, but also for so far non-treatable, genetically-mediated disorders of the nervous system accompanied by pathogenic neuroinflammation.

Altered motor plasticity in an acute relapse of multiple sclerosis.

In relapsing-remitting MS (RRMS), the symptoms of a clinical relapse subside over time. Neuroplasticity is believed to play an important compensatory role. In this study, we assessed excitability-decreasing plasticity during an acute relapse of MS and 12 weeks afterwards. Motor plasticity was examined in 19 patients with clinically isolated syndrome or RRMS during a steroid-treated relapse (t1) and 12 weeks afterwards (t2) using paired-associative stimulation (PAS10). This method combines repetitive electric nerve stimulation with transcranial magnetic stimulation of the contralateral motor cortex to model long-term synaptic depression in the human cortex. Additionally, 19 age-matched healthy controls were assessed. Motor-evoked potentials of the abductor pollicis brevis muscle were recorded before and after intervention. Clinical disability was assessed by the multiple sclerosis functional composite and the subscore of the nine-hole peg test taken as a measure of hand function. The effect of PAS10 was significantly different between controls and patients; at t1, but not at t2, baseline-normalized postinterventional amplitudes were significantly higher in patients (106 [IQR 98-137] % post10-15 and 111 [IQR 88-133] % post20-25) compared to controls (92 [IQR 85-111] % and 90 [IQR 75-102] %). Additional exploratory analysis indicated a potentially excitability-enhancing effect of PAS10 in patients as opposed to controls. Significant clinical improvement between t1 and t2 was not correlated with PAS10 effects. Our results indicate an alteration of PAS10-induced synaptic plasticity during relapse, presumably reflecting a polarity shift due to metaplastic processes within the motor cortex. Further studies will need to elucidate the functional significance of such changes for the clinical course of MS.

Novel insights into neuroinflammation: bacterial lipopolysaccharide, tumor necrosis factor α, and Ureaplasma species differentially modulate atypical chemokine receptor 3 responses in human brain microvascular endothelial cells.

BACKGROUND: Atypical chemokine receptor 3 (ACKR3, synonym CXCR7) is increasingly considered relevant in neuroinflammatory conditions, in which its upregulation contributes to compromised endothelial barrier function and may ultimately allow inflammatory brain injury. While an impact of ACKR3 has been recognized in several neurological autoimmune diseases, neuroinflammation may also result from infectious agents, including Ureaplasma species (spp.). Although commonly regarded as commensals of the adult urogenital tract, Ureaplasma spp. may cause invasive infections in immunocompromised adults as well as in neonates and appear to be relevant pathogens in neonatal meningitis. Nonetheless, clinical and in vitro data on Ureaplasma-induced inflammation are scarce. METHODS: We established a cell culture model of Ureaplasma meningitis, aiming to analyze ACKR3 variances as a possible pathomechanism in Ureaplasma-associated neuroinflammation. Non-immortalized human brain microvascular endothelial cells (HBMEC) were exposed to bacterial lipopolysaccharide (LPS) or tumor necrosis factor-α (TNF-α), and native as well as LPS-primed HBMEC were cultured with Ureaplasma urealyticum serovar 8 (Uu8) and U. parvum serovar 3 (Up3). ACKR3 responses were assessed via qRT-PCR, RNA sequencing, flow cytometry, and immunocytochemistry. RESULTS: LPS, TNF-α, and Ureaplasma spp. influenced ACKR3 expression in HBMEC. LPS and TNF-α significantly induced ACKR3 mRNA expression (p < 0.001, vs. control), whereas Ureaplasma spp. enhanced ACKR3 protein expression in HBMEC (p < 0.01, vs. broth control). Co-stimulation with LPS and either Ureaplasma isolate intensified ACKR3 responses (p < 0.05, vs. LPS). Furthermore, stimulation wielded a differential influence on the receptor's ligands. CONCLUSIONS: We introduce an in vitro model of Ureaplasma meningitis. We are able to demonstrate a pro-inflammatory capacity of Ureaplasma spp. in native and, even more so, in LPS-primed HBMEC, underlining their clinical relevance particularly in a setting of co-infection. Furthermore, our data may indicate a novel role for ACKR3, with an impact not limited to auto-inflammatory diseases, but extending to infection-related neuroinflammation as well. AKCR3-induced blood-brain barrier breakdown might constitute a potential common pathomechanism.

Auto-antibodies to contactin-associated protein 1 (Caspr) in two patients with painful inflammatory neuropathy.

Auto-antibodies against the paranodal proteins neurofascin-155 and contactin-1 have recently been described in patients with chronic inflammatory demyelinating polyradiculoneuropathy and are associated with a distinct clinical phenotype and response to treatment. Contactin-associated protein 1 (Caspr, encoded by CNTNAP1) is a paranodal protein that is attached to neurofascin-155 and contactin-1 (CNTN1) but has not yet been identified as a sole antigen in patients with inflammatory neuropathies. In the present study, we screened a cohort of 35 patients with chronic inflammatory demyelinating polyradiculoneuropathy (age range 20-80, 10 female, 25 male) and 22 patients with Guillain-Barré syndrome (age range 17-86, eight female, 14 male) for autoantibodies against paranodal antigens. We identified two patients, one with chronic inflammatory demyelinating polyradiculoneuropathy and one with Guillain-Barré syndrome, with autoantibodies against Caspr by binding assays using Caspr transfected human embryonic kidney cells and murine teased fibres. IgG3 was the predominant autoantibody subclass in the patient with Guillain-Barré syndrome, IgG4 was predominant in the patient with chronic inflammatory demyelinating polyradiculoneuropathy. Accordingly, complement deposition after binding to HEK293 cells was detectable in the patient with IgG3 autoantibodies only, not in the patient with IgG4. Severe disruption of the paranodal and nodal architecture was detectable in teased fibres of the sural nerve biopsy and in dermal myelinated fibres, supporting the notion of the paranodes being the site of pathology. Deposition of IgG at the paranodes was detected in teased fibre preparations of the sural nerve, further supporting the pathogenicity of anti-Caspr autoantibodies. Pain was one of the predominant findings in both patients, possibly reflected by binding of patients' IgG to TRPV1 immunoreactive dorsal root ganglia neurons. Our results demonstrate that the paranodal protein Caspr constitutes a new antigen that leads to autoantibody generation as part of the novel entity of neuropathies associated with autoantibodies against paranodal proteins.

NFAT1 deficit and NFAT2 deficit attenuate EAE via different mechanisms.

EAE serves as an animal model for multiple sclerosis and is initiated by autoreactive T cells that infiltrate the CNS. Recognition of myelin-associated Ags within the CNS leads to activation of the transcription factor family NFAT. Here, we demonstrate an essential role for NFAT in disease induction, as the combined lack of NFAT1 (NFATc2) and NFAT2 (NFATc1) completely protected mice. Single deficiency of either NFAT1 or NFAT2 ameliorated the course of EAE, and NFAT2 ablation resulted in an obstructed proinflammatory reaction. However, NFAT1 deficit led to an anti-inflammatory response with nonpathogenic Th17 and Th2 cells concurrently secreting IL-17, IL-4, and IL-10. Both IL-4 and IL-10 contributed to disease protection. In Nfat1(-/-) CD4(+) T cells, the expression of anti-inflammatory lymphokines was mediated by NFAT2, thus directly enabling protective IL expression. Consequently, blocking NFAT in toto may be an option for immunosuppressive therapy. More importantly, selective NFAT1 blockade could represent a safe long-term immunomodulatory treatment approach for multiple sclerosis patients, potentially avoiding the adverse effects of global immunosuppression.

MOG-IgG in NMO and related disorders: a multicenter study of 50 patients. Part 4: Afferent visual system damage after optic neuritis in MOG-IgG-seropositive versus AQP4-IgG-seropositive patients.

BACKGROUND: Antibodies against myelin oligodendrocyte glycoprotein (MOG-IgG) have been reported in patients with aquaporin-4 antibody (AQP4-IgG)-negative neuromyelitis optica spectrum disorders (NMOSD). The objective of this study was to describe optic neuritis (ON)-induced neuro-axonal damage in the retina of MOG-IgG-positive patients in comparison with AQP4-IgG-positive NMOSD patients. METHODS: Afferent visual system damage following ON was bilaterally assessed in 16 MOG-IgG-positive patients with a history of ON and compared with that in 16 AQP4-IgG-positive NMOSD patients. In addition, 16 healthy controls matched for age, sex, and disease duration were analyzed. Study data included ON history, retinal optical coherence tomography, visual acuity, and visual evoked potentials. RESULTS: Eight MOG-IgG-positive patients had a previous diagnosis of AQP4-IgG-negative NMOSD with ON and myelitis, and eight of (mainly recurrent) ON. Twenty-nine of the 32 eyes of the MOG-IgG-positive patients had been affected by at least one episode of ON. Peripapillary retinal nerve fiber layer thickness (pRNFL) and ganglion cell and inner plexiform layer volume (GCIP) were significantly reduced in ON eyes of MOG-IgG-positive patients (pRNFL = 59 ± 23 µm; GCIP = 1.50 ± 0.34 mm3) compared with healthy controls (pRNFL = 99 ± 6 µm, p < 0.001; GCIP = 1.97 ± 0.11 mm3, p < 0.001). Visual acuity was impaired in eyes after ON in MOG-IgG-positive patients (0.35 ± 0.88 logMAR). There were no significant differences in any structural or functional visual parameters between MOG-IgG-positive and AQP4-IgG-positive patients (pRNFL: 59 ± 21 µm; GCIP: 1.41 ± 0.27 mm3; Visual acuity = 0.72 ± 1.09 logMAR). Importantly, MOG-IgG-positive patients had a significantly higher annual ON relapse rate than AQP4-IgG-positive patients (median 0.69 vs. 0.29 attacks/year, p = 0.004), meaning that on average a single ON episode caused less damage in MOG-IgG-positive than in AQP4-IgG-positive patients. pRNFL and GCIP loss correlated with the number of ON episodes in MOG-IgG-positive patients (p < 0.001), but not in AQP4-IgG-positive patients. CONCLUSIONS: Retinal neuro-axonal damage and visual impairment after ON in MOG-IgG-positive patients are as severe as in AQP4-IgG-positive NMOSD patients. In MOG-IgG-positive patients, damage accrual may be driven by higher relapse rates, whereas AQP4-IgG-positive patients showed fewer but more severe episodes of ON. Given the marked damage in some of our MOG-IgG-positive patients, early diagnosis and timely initiation and close monitoring of immunosuppressive therapy are important.

MOG-IgG in NMO and related disorders: a multicenter study of 50 patients. Part 2: Epidemiology, clinical presentation, radiological and laboratory features, treatment responses, and long-term outcome.

BACKGROUND: A subset of patients with neuromyelitis optica spectrum disorders (NMOSD) has been shown to be seropositive for myelin oligodendrocyte glycoprotein antibodies (MOG-IgG). OBJECTIVE: To describe the epidemiological, clinical, radiological, cerebrospinal fluid (CSF), and electrophysiological features of a large cohort of MOG-IgG-positive patients with optic neuritis (ON) and/or myelitis (n = 50) as well as attack and long-term treatment outcomes. METHODS: Retrospective multicenter study. RESULTS: The sex ratio was 1:2.8 (m:f). Median age at onset was 31 years (range 6-70). The disease followed a multiphasic course in 80 % (median time-to-first-relapse 5 months; annualized relapse rate 0.92) and resulted in significant disability in 40 % (mean follow-up 75 ± 46.5 months), with severe visual impairment or functional blindness (36 %) and markedly impaired ambulation due to paresis or ataxia (25 %) as the most common long-term sequelae. Functional blindess in one or both eyes was noted during at least one ON attack in around 70 %. Perioptic enhancement was present in several patients. Besides acute tetra-/paraparesis, dysesthesia and pain were common in acute myelitis (70 %). Longitudinally extensive spinal cord lesions were frequent, but short lesions occurred at least once in 44 %. Fourty-one percent had a history of simultaneous ON and myelitis. Clinical or radiological involvement of the brain, brainstem, or cerebellum was present in 50 %; extra-opticospinal symptoms included intractable nausea and vomiting and respiratory insufficiency (fatal in one). CSF pleocytosis (partly neutrophilic) was present in 70 %, oligoclonal bands in only 13 %, and blood-CSF-barrier dysfunction in 32 %. Intravenous methylprednisolone (IVMP) and long-term immunosuppression were often effective; however, treatment failure leading to rapid accumulation of disability was noted in many patients as well as flare-ups after steroid withdrawal. Full recovery was achieved by plasma exchange in some cases, including after IVMP failure. Breakthrough attacks under azathioprine were linked to the drug-specific latency period and a lack of cotreatment with oral steroids. Methotrexate was effective in 5/6 patients. Interferon-beta was associated with ongoing or increasing disease activity. Rituximab and ofatumumab were effective in some patients. However, treatment with rituximab was followed by early relapses in several cases; end-of-dose relapses occurred 9-12 months after the first infusion. Coexisting autoimmunity was rare (9 %). Wingerchuk's 2006 and 2015 criteria for NMO(SD) and Barkhof and McDonald criteria for multiple sclerosis (MS) were met by 28 %, 32 %, 15 %, 33 %, respectively; MS had been suspected in 36 %. Disease onset or relapses were preceded by infection, vaccination, or pregnancy/delivery in several cases. CONCLUSION: Our findings from a predominantly Caucasian cohort strongly argue against the concept of MOG-IgG denoting a mild and usually monophasic variant of NMOSD. The predominantly relapsing and often severe disease course and the short median time to second attack support the use of prophylactic long-term treatments in patients with MOG-IgG-positive ON and/or myelitis.

MOG-IgG in NMO and related disorders: a multicenter study of 50 patients. Part 1: Frequency, syndrome specificity, influence of disease activity, long-term course, association with AQP4-IgG, and origin.

BACKGROUND: Antibodies to myelin oligodendrocyte glycoprotein (MOG-IgG) have been suggested to play a role in a subset of patients with neuromyelitis optica and related disorders. OBJECTIVE: To assess (i) the frequency of MOG-IgG in a large and predominantly Caucasian cohort of patients with optic neuritis (ON) and/or myelitis; (ii) the frequency of MOG-IgG among AQP4-IgG-positive patients and vice versa; (iii) the origin and frequency of MOG-IgG in the cerebrospinal fluid (CSF); (iv) the presence of MOG-IgG at disease onset; and (v) the influence of disease activity and treatment status on MOG-IgG titers. METHODS: 614 serum samples from patients with ON and/or myelitis and from controls, including 92 follow-up samples from 55 subjects, and 18 CSF samples were tested for MOG-IgG using a live cell-based assay (CBA) employing full-length human MOG-transfected HEK293A cells. RESULTS: MOG-IgG was detected in 95 sera from 50 patients with ON and/or myelitis, including 22/54 (40.7 %) patients with a history of both ON and myelitis, 22/103 (21.4 %) with a history of ON but no myelitis and 6/45 (13.3 %) with a history of longitudinally extensive transverse myelitis but no ON, and in 1 control patient with encephalitis and a connective tissue disorder, all of whom were negative for AQP4-IgG. MOG-IgG was absent in 221 further controls, including 83 patients with AQP4-IgG-seropositive neuromyelitis optica spectrum disorders and 85 with multiple sclerosis (MS). MOG-IgG was found in 12/18 (67 %) CSF samples from MOG-IgG-seropositive patients; the MOG-IgG-specific antibody index was negative in all cases, indicating a predominantly peripheral origin of CSF MOG-IgG. Serum and CSF MOG-IgG belonged to the complement-activating IgG1 subclass. MOG-IgG was present already at disease onset. The antibodies remained detectable in 40/45 (89 %) follow-up samples obtained over a median period of 16.5 months (range 0-123). Serum titers were higher during attacks than during remission (p < 0.0001), highest during attacks of simultaneous myelitis and ON, lowest during acute isolated ON, and declined following treatment. CONCLUSIONS: To date, this is the largest cohort studied for IgG to human full-length MOG by means of an up-to-date CBA. MOG-IgG is present in a substantial subset of patients with ON and/or myelitis, but not in classical MS. Co-existence of MOG-IgG and AQP4-IgG is highly uncommon. CSF MOG-IgG is of extrathecal origin. Serum MOG-IgG is present already at disease onset and remains detectable in the long-term course. Serum titers depend on disease activity and treatment status.

MOG-IgG in NMO and related disorders: a multicenter study of 50 patients. Part 3: Brainstem involvement - frequency, presentation and outcome.

BACKGROUND: Myelin oligodendrocyte glycoprotein antibodies (MOG-IgG) are present in a subset of aquaporin-4 (AQP4)-IgG-negative patients with optic neuritis (ON) and/or myelitis. Little is known so far about brainstem involvement in MOG-IgG-positive patients. OBJECTIVE: To investigate the frequency, clinical and paraclinical features, course, outcome, and prognostic implications of brainstem involvement in MOG-IgG-positive ON and/or myelitis. METHODS: Retrospective case study. RESULTS: Among 50 patients with MOG-IgG-positive ON and/or myelitis, 15 (30 %) with a history of brainstem encephalitis were identified. All were negative for AQP4-IgG. Symptoms included respiratory insufficiency, intractable nausea and vomiting (INV), dysarthria, dysphagia, impaired cough reflex, oculomotor nerve palsy and diplopia, nystagmus, internuclear ophthalmoplegia (INO), facial nerve paresis, trigeminal hypesthesia/dysesthesia, vertigo, hearing loss, balance difficulties, and gait and limb ataxia; brainstem involvement was asymptomatic in three cases. Brainstem inflammation was already present at or very shortly after disease onset in 7/15 (47 %) patients. 16/21 (76.2 %) brainstem attacks were accompanied by acute myelitis and/or ON. Lesions were located in the pons (11/13), medulla oblongata (8/14), mesencephalon (cerebral peduncles; 2/14), and cerebellar peduncles (5/14), were adjacent to the fourth ventricle in 2/12, and periaqueductal in 1/12; some had concomitant diencephalic (2/13) or cerebellar lesions (1/14). MRI or laboratory signs of blood-brain barrier damage were present in 5/12. Cerebrospinal fluid pleocytosis was found in 11/14 cases, with neutrophils in 7/11 (3-34 % of all CSF white blood cells), and oligoclonal bands in 4/14. Attacks were preceded by acute infection or vaccination in 5/15 (33.3 %). A history of teratoma was noted in one case. The disease followed a relapsing course in 13/15 (87 %); the brainstem was involved more than once in 6. Immunosuppression was not always effective in preventing relapses. Interferon-beta was followed by new attacks in two patients. While one patient died from central hypoventilation, partial or complete recovery was achieved in the remainder following treatment with high-dose steroids and/or plasma exchange. Brainstem involvement was associated with a more aggressive general disease course (higher relapse rate, more myelitis attacks, more frequently supratentorial brain lesions, worse EDSS at last follow-up). CONCLUSIONS: Brainstem involvement is present in around one third of MOG-IgG-positive patients with ON and/or myelitis. Clinical manifestations are diverse and may include symptoms typically seen in AQP4-IgG-positive neuromyelitis optica, such as INV and respiratory insufficiency, or in multiple sclerosis, such as INO. As MOG-IgG-positive brainstem encephalitis may take a serious or even fatal course, particular attention should be paid to signs or symptoms of additional brainstem involvement in patients presenting with MOG-IgG-positive ON and/or myelitis.

Oligoclonal bands predict multiple sclerosis in children with optic neuritis.

We retrospectively evaluated predictors of conversion to multiple sclerosis (MS) in 357 children with isolated optic neuritis (ON) as a first demyelinating event who had a median follow-up of 4.0 years. Multiple Cox proportional-hazards regressions revealed abnormal cranial magnet resonance imaging (cMRI; hazard ratio [HR] = 5.94, 95% confidence interval [CI] = 3.39-10.39, p < 0.001), presence of cerebrospinal fluid immunoglobulin G oligoclonal bands (OCB; HR = 3.69, 95% CI = 2.32-5.86, p < 0.001), and age (HR = 1.08 per year of age, 95% CI = 1.02-1.13, p = 0.003) as independent predictors of conversion, whereas sex and laterality (unilateral vs bilateral) had no influence. Combined cMRI and OCB positivity indicated a 26.84-fold higher HR for developing MS compared to double negativity (95% CI = 12.26-58.74, p < 0.001). Accordingly, cerebrospinal fluid analysis may supplement cMRI to determine the risk of MS in children with isolated ON.

Genome-wide significant association with seven novel multiple sclerosis risk loci.

OBJECTIVE: A recent large-scale study in multiple sclerosis (MS) using the ImmunoChip platform reported on 11 loci that showed suggestive genetic association with MS. Additional data in sufficiently sized and independent data sets are needed to assess whether these loci represent genuine MS risk factors. METHODS: The lead SNPs of all 11 loci were genotyped in 10 796 MS cases and 10 793 controls from Germany, Spain, France, the Netherlands, Austria and Russia, that were independent from the previously reported cohorts. Association analyses were performed using logistic regression based on an additive model. Summary effect size estimates were calculated using fixed-effect meta-analysis. RESULTS: Seven of the 11 tested SNPs showed significant association with MS susceptibility in the 21 589 individuals analysed here. Meta-analysis across our and previously published MS case-control data (total sample size n=101 683) revealed novel genome-wide significant association with MS susceptibility (p<5×10(-8)) for all seven variants. This included SNPs in or near LOC100506457 (rs1534422, p=4.03×10(-12)), CD28 (rs6435203, p=1.35×10(-9)), LPP (rs4686953, p=3.35×10(-8)), ETS1 (rs3809006, p=7.74×10(-9)), DLEU1 (rs806349, p=8.14×10(-12)), LPIN3 (rs6072343, p=7.16×10(-12)) and IFNGR2 (rs9808753, p=4.40×10(-10)). Cis expression quantitative locus effects were observed in silico for rs6435203 on CD28 and for rs9808753 on several immunologically relevant genes in the IFNGR2 locus. CONCLUSIONS: This study adds seven loci to the list of genuine MS genetic risk factors and further extends the list of established loci shared across autoimmune diseases.

Soluble VCAM-1 impairs human brain endothelial barrier integrity via integrin α-4-transduced outside-in signalling.

Human brain microvascular endothelial cells forming the blood-brain barrier (BBB) release soluble vascular cell adhesion molecule-1 (sVCAM-1) under inflammatory conditions. Furthermore, sVCAM-1 serum levels in untreated patients with multiple sclerosis (MS) correlate with a breakdown of the BBB as measured by gadolinium-enhanced MRI. To date, it is unknown whether sVCAM-1 itself modulates BBB permeability. Here, we provide evidence that human brain endothelium expresses integrin α-4/ß-1, the molecular binding partner of sVCAM-1, and that sVCAM-1 directly impairs BBB function by inducing intracellular signalling events through integrin α-4. Primary human brain microvascular endothelial cells showed low to moderate integrin α-4 and strong ß-1 but no definite ß-7 expression in vitro and in situ. Increased brain endothelial integrin α-4 expression was observed in active MS lesions in situ and after angiogenic stimulation in vitro. Exposure of cultured primary brain endothelial cells to recombinant sVCAM-1 significantly increased their permeability to the soluble tracer dextran, which was paralleled by formation of actin stress fibres and reduced staining of tight junction-associated molecules. Soluble VCAM-1 was also found to activate Rho GTPase and p38 MAP kinase. Chemical inhibition of these signalling pathways partially prevented sVCAM-1-induced changes of tight junction arrangement. Importantly, natalizumab, a neutralising recombinant monoclonal antibody against integrin α-4 approved for the treatment of patients with relapsing-remitting MS, partially antagonised the barrier-disturbing effect of sVCAM-1. In summary, we newly characterised sVCAM-1 as a compromising factor of brain endothelial barrier function that may be partially blocked by the MS therapeutic natalizumab.

Fumaric Acid Esters Do Not Reduce Inflammatory NF-κB/p65 Nuclear Translocation, ICAM-1 Expression and T-Cell Adhesiveness of Human Brain Microvascular Endothelial Cells.

Dimethyl fumarate (DMF) is approved for disease-modifying treatment of patients with relapsing-remitting multiple sclerosis. Animal experiments suggested that part of its therapeutic effect is due to a reduction of T-cell infiltration of the central nervous system (CNS) by uncertain mechanisms. Here we evaluated whether DMF and its primary metabolite monomethyl fumarate (MMF) modulate pro-inflammatory intracellular signaling and T-cell adhesiveness of nonimmortalized single donor human brain microvascular endothelial cells at low passages. Neither DMF nor MMF at concentrations of 10 or 50 µM blocked the IL-1ß-induced nuclear translocation of NF-κB/p65, whereas the higher concentration of DMF inhibited the nuclear entry of p65 in human umbilical vein endothelium cultured in parallel. DMF and MMF also did not alter the IL-1ß-stimulated activation of p38 MAPK in brain endothelium. Furthermore, neither DMF nor MMF reduced the basal or IL-1ß-inducible expression of ICAM-1. In accordance, both fumaric acid esters did not reduce the adhesion of activated Jurkat T cells to brain endothelium under basal or inflammatory conditions. Therefore, brain endothelial cells probably do not directly mediate a potential blocking effect of fumaric acid esters on the inflammatory infiltration of the CNS by T cells.

Assessment of microRNA-related SNP effects in the 3' untranslated region of the IL22RA2 risk locus in multiple sclerosis.

Recent large-scale association studies have identified over 100 MS risk loci. One of these MS risk variants is single-nucleotide polymorphism (SNP) rs17066096, located ~14 kb downstream of IL22RA2. IL22RA2 represents a compelling MS candidate gene due to the role of IL-22 in autoimmunity; however, rs17066096 does not map into any known functional element. We assessed whether rs17066096 or a nearby proxy SNP may exert pathogenic effects by affecting microRNA-to-mRNA binding and thus IL22RA2 expression using comprehensive in silico predictions, in vitro reporter assays, and genotyping experiments in 6,722 individuals. In silico screening identified two predicted microRNA binding sites in the 3'UTR of IL22RA2 (for hsa-miR-2278 and hsa-miR-411-5p) encompassing a SNP (rs28366) in moderate linkage disequilibrium with rs17066096 (r (2) = 0.4). The binding of both microRNAs to the IL22RA2 3'UTR was confirmed in vitro, but their binding affinities were not significantly affected by rs28366. Association analyses revealed significant association of rs17066096 and MS risk in our independent German dataset (odds ratio = 1.15, P = 3.48 × 10(-4)), but did not indicate rs28366 to be the cause of this signal. While our study provides independent validation of the association between rs17066096 and MS risk, this signal does not appear to be caused by sequence variants affecting microRNA function.

MANBA, CXCR5, SOX8, RPS6KB1 and ZBTB46 are genetic risk loci for multiple sclerosis.

A recent genome-wide association study reported five loci for which there was strong, but sub-genome-wide significant evidence for association with multiple sclerosis risk. The aim of this study was to evaluate the role of these potential risk loci in a large and independent data set of ≈ 20,000 subjects. We tested five single nucleotide polymorphisms rs228614 (MANBA), rs630923 (CXCR5), rs2744148 (SOX8), rs180515 (RPS6KB1), and rs6062314 (ZBTB46) for association with multiple sclerosis risk in a total of 8499 cases with multiple sclerosis, 8765 unrelated control subjects and 958 trios of European descent. In addition, we assessed the overall evidence for association by combining these newly generated data with the results from the original genome-wide association study by meta-analysis. All five tested single nucleotide polymorphisms showed consistent and statistically significant evidence for association with multiple sclerosis in our validation data sets (rs228614: odds ratio = 0.91, P = 2.4 × 10(-6); rs630923: odds ratio = 0.89, P = 1.2 × 10(-4); rs2744148: odds ratio = 1.14, P = 1.8 × 10(-6); rs180515: odds ratio = 1.12, P = 5.2 × 10(-7); rs6062314: odds ratio = 0.90, P = 4.3 × 10(-3)). Combining our data with results from the previous genome-wide association study by meta-analysis, the evidence for association was strengthened further, surpassing the threshold for genome-wide significance (P < 5 × 10(-8)) in each case. Our study provides compelling evidence that these five loci are genuine multiple sclerosis susceptibility loci. These results may eventually lead to a better understanding of the underlying disease pathophysiology.

OzEAN Study to Collect Real-World Evidence of Persistent Use, Effectiveness, and Safety of Ozanimod Over 5 Years in Patients With Relapsing-Remitting Multiple Sclerosis in Germany.

Background: Ozanimod, a sphingosine 1-phosphate receptor 1 and 5 modulator, was approved as a disease-modifying therapy for active relapsing-remitting multiple sclerosis (RRMS) in 2020 and for active ulcerative colitis in 2021. Long-term, real-world studies in a nonselective population are needed. OzEAN is an ongoing study to assess the real-world persistent use, effectiveness, and safety of ozanimod and its impact on quality of life (QoL) in patients with RRMS over a 5-year period. Methods: This prospective, noninterventional, postmarketing authorization study will enroll ~1,300 patients (≥18 years of age) with active RRMS. The decision to initiate ozanimod must have been made before and independent from study participation. Enrollment began in March 2021. Recruitment is ongoing and will last for 36 months across 140 sites in Germany. Treatment-naive patients or those having prior experience with a disease-modifying therapy receive oral ozanimod 0.92 mg/day after an initial dose escalation, per the summary of product characteristics recommendations, for up to 60 months. Persistence with ozanimod treatment (primary endpoint) is assessed at month 60. Secondary endpoints include additional physician-reported outcomes [persistence at earlier time points, annualized relapse rate, Expanded Disability Status Scale score, cognition (Symbol Digit Modalities Test), and incidence of adverse events], and patient-reported outcomes assessing patient satisfaction, adherence, and treatment modalities (Treatment Satisfaction Questionnaire for Medication, v1.4), disability (United Kingdom Neurological Disability Rating Scale), QoL (MSQOL-54 questionnaire), fatigue (Fatigue Scale for Motor and Cognitive Functions), and health economics [Work Productivity and Activity Impairment Questionnaire for Multiple Sclerosis (German v2.1); Multiple Sclerosis Health Resource Survey, v3.0]. A Multiple Sclerosis Documentation System with an internet-based e-health portal allows patients to view files and complete questionnaires. A safety follow-up will occur 3-8 months after the last ozanimod dose for patients who discontinue treatment early. Long-term results are anticipated after study completion in 2029. Yearly interim analyses are planned after enrollment has reached 25%. Conclusion: This is the first long-term, real-world study of ozanimod in patients with RRMS and, to our knowledge, the first noninterventional study utilizing a patient portal. These data will add to the safety/efficacy profile of ozanimod demonstrated in phase 3 trials. Clinical Trial Registration: Clinicaltrials.gov, identifier: NCT05335031.

Safety, Adherence and Persistence in a Real-World Cohort of German MS Patients Newly Treated With Ocrelizumab: First Insights From the CONFIDENCE Study.

Background: Real-world relapsing multiple sclerosis (RMS) and primary progressive MS (PPMS) populations may be more diverse than in clinical trials. Here, we present a first analysis of safety, adherence and persistence data from a real-world cohort of patients newly treated with ocrelizumab. Methods: CONFIDENCE (ML39632, EUPAS22951) is an ongoing multicenter, non-interventional post authorization safety study assessing patients with RMS or PPMS newly treated with ocrelizumab or other disease-modifying therapies for up to 10 years. For this analysis, patients newly treated with ocrelizumab were analyzed in subgroups by MS phenotype and age over a mean ~1 year of exposure totaling 2,329 patient years [PY]). Results: At data cutoff (14 October 2020), 1,702 patients with RMS and 398 patients with PPMS were treated with ≥1 dose of ocrelizumab. At baseline, the mean ages (SD) of patients with RMS and PPMS were 41.59 (11.24) and 50.95 (9.88) years and the mean EDSS (Expanded Disability Status Scale) was 3.18 (1.87) and 4.41 (1.59), respectively. The most common adverse events (AEs) and serious AEs across both phenotypes were infections and infestations, with infection SAE rates of 2.8 events/100 PY and 1.5 events/100 PY in patients with RMS and PPMS, respectively. Across all phenotypes, ocrelizumab persistence was 92% at 24 months; median time between doses was ~6 months. Conclusions: The ocrelizumab safety profile observed in the CONFIDENCE real-world MS population was consistent to the one observed in pivotal clinical trials. High treatment persistence and adherence were observed. Trial Registration: ML39632, EUPAS22951.