Ocrelizumab B cell depletion has no effect on HERV RNA expression in PBMC in MS patients.

BACKGROUND: Epstein barr virus (EBV) infection of B cells is now understood to be one of the triggering events for the development of Multiple Sclerosis (MS), a progressive immune-mediated disease of the central nervous system. EBV infection is also linked to expression of human endogenous retroviruses (HERVs) of the HERV-W group, a further risk factor for the development of MS. Ocrelizumab is a high-potency disease-modifying treatment (DMT) for MS, which depletes B cells by targeting CD20. OBJECTIVES: We studied the effects of ocrelizumab on gene expression in peripheral blood mononuclear cells (PBMC) from paired samples from 20 patients taken prior to and 6 months after beginning ocrelizumab therapy. We hypothesised that EBV and HERV-W loads would be lower in post-treatment samples. METHODS: Samples were collected in Paxgene tubes, subject to RNA extraction and Illumina paired end short read mRNA sequencing with mapping of sequence reads to the human genome using Salmon and differential gene expression compared with DeSeq2. Mapping was also performed separately to the HERV-D database of HERV sequences and the EBV reference sequence. RESULTS: Patient samples were more strongly clustered by individual rather than disease type (relapsing/remitting or primary progressive), treatment (pre and post), age, or sex. Fourteen genes, all clearly linked to B cell function were significantly down regulated in the post treatment samples. Interestingly only one pre-treatment sample had detectable EBV RNA and there were no significant differences in HERV expression (of any group) between pre- and post-treatment samples. CONCLUSIONS: While EBV and HERV expression are clearly linked to triggering MS pathogenesis, it does not appear that high level expression of these viruses is a part of the ongoing disease process or that changes in virus load are associated with ocrelizumab treatment.

Sjogren's myelitis presenting as hemicord syndrome.

We present a case where a 63-year-old right-handed man who presented with a 6-month history of progressive asymmetrical sensorimotor symptoms in lower limbs. This was associated with concomitant rash on the lower limbs, and mild sicca symptoms. MRI spine showed focal T2 hyperintensity in the left hemicord at C3-4 level. Skin biopsy of the rash revealed urticarial vasculitis, and lip biopsy revealed lymphocytic sialadenitis. Initial anti-Ro antibody was negative, but subsequent Ro52 antibody testing returned positive. There was also matched serum and cerebrospinal fluid oligoclonal bands. He was subsequently diagnosed as Sjogren's myelitis and treated with intravenous methylprednisolone, then transitioned to a steroid sparing agent. This case highlights the difficulties in reaching a rheumatological diagnosis in the early stages with typical negative antibodies, and shows a rare neurological manifestation of a systemic rheumatological condition.

ADAMS project: a genetic Association study in individuals from Diverse Ancestral backgrounds with Multiple Sclerosis based in the UK.

PURPOSE: Genetic studies of multiple sclerosis (MS) susceptibility and severity have focused on populations of European ancestry. Studying MS genetics in other ancestral groups is necessary to determine the generalisability of these findings. The genetic Association study in individuals from Diverse Ancestral backgrounds with Multiple Sclerosis (ADAMS) project aims to gather genetic and phenotypic data on a large cohort of ancestrally-diverse individuals with MS living in the UK. PARTICIPANTS: Adults with self-reported MS from diverse ancestral backgrounds. Recruitment is via clinical sites, online (https://app.mantal.co.uk/adams) or the UK MS Register. We are collecting demographic and phenotypic data using a baseline questionnaire and subsequent healthcare record linkage. We are collecting DNA from participants using saliva kits (Oragene-600) and genotyping using the Illumina Global Screening Array V.3. FINDINGS TO DATE: As of 3 January 2023, we have recruited 682 participants (n=446 online, n=55 via sites, n=181 via the UK MS Register). Of this initial cohort, 71.2% of participants are female, with a median age of 44.9 years at recruitment. Over 60% of the cohort are non-white British, with 23.5% identifying as Asian or Asian British, 16.2% as Black, African, Caribbean or Black British and 20.9% identifying as having mixed or other backgrounds. The median age at first symptom is 28 years, and median age at diagnosis is 32 years. 76.8% have relapsing-remitting MS, and 13.5% have secondary progressive MS. FUTURE PLANS: Recruitment will continue over the next 10 years. Genotyping and genetic data quality control are ongoing. Within the next 3 years, we aim to perform initial genetic analyses of susceptibility and severity with a view to replicating the findings from European-ancestry studies. In the long term, genetic data will be combined with other datasets to further cross-ancestry genetic discoveries.

Investigating the Effect of Cigarette Smoking on Serum Uric Acid Levels in Multiple Sclerosis Patients: A Cross Sectional Study.

OBJECTIVES: The present study is aimed at determining the effect of cigarette smoking (CS) on serum uric acid (UA) levels quantitatively before and after smoking cessation among people with MS (pwMS). Additionally, a possible correlation between UA levels and both disability progression and disease severity was also investigated. A retrospective cross-sectional study was conducted using the Nottingham University Hospitals MS Clinics database. It involves 127 people with definite MS recorded when reporting the latest smoking status and the clinical diagnosis. All necessary demographics and clinical characteristics were collected. We found that smoker pwMS had significantly lower serum UA levels than non-smoker pwMS (p-value = 0.0475), and this reduction was recovered after smoking cessation (p-value = 0.0216). However, the levels of disability or disease severity were not correlated with the levels of serum UA in current smoker pwMS, measured by the expanded disability status scale (EDSS; r = -0.24; p-value = 0.38), multiple sclerosis impact scale 29 (MSIS-29; r = 0.01; p-value = 0.97) and MS severity score (MSSS; r = -0.16; p-value = 0.58), respectively. Our result suggests that the reduction in UA levels is more likely a consequence of oxidative stress triggered by many risk factors, including CS, and could be considered a potential indicator of smoking cessation. In addition, the absence of a correlation between UA levels and disease severity and disability suggests that UA is not an optimal biomarker for disease severity and disability prediction among current smoker, ex-smoker or non-smoker pwMS.

Sjogren's myelitis presenting as hemicord syndrome.

We present a case where a 63-year-old right-handed man who presented with a 6-month history of progressive asymmetrical sensorimotor symptoms in lower limbs. This was associated with concomitant rash on the lower limbs, and mild sicca symptoms. MRI spine showed focal T2 hyperintensity in the left hemicord at C3-4 level. Skin biopsy of the rash revealed urticarial vasculitis, and lip biopsy revealed lymphocytic sialadenitis. Initial anti-Ro antibody was negative, but subsequent Ro52 antibody testing returned positive. There was also matched serum and cerebrospinal fluid oligoclonal bands. He was subsequently diagnosed as Sjogren's myelitis and treated with intravenous methylprednisolone, then transitioned to a steroid sparing agent. This case highlights the difficulties in reaching a rheumatological diagnosis in the early stages with typical negative antibodies, and shows a rare neurological manifestation of a systemic rheumatological condition.

Multiple sclerosis and the risk of infection: Association of British Neurologists consensus guideline.

Infection in people with multiple sclerosis (MS) is of major concern, particularly for those receiving disease-modifying therapies. This article explores the risk of infection in people with MS and provides guidance-developed by Delphi consensus by specialists involved in their management-on how to screen for, prevent and manage infection in this population.

The Potential for EBV Vaccines to Prevent Multiple Sclerosis.

There is increasing evidence suggesting that Epstein-Barr virus infection is a causative factor of multiple sclerosis (MS). Epstein-Barr virus (EBV) is a human herpesvirus, Human Gammaherpesvirus 4. EBV infection shows two peaks: firstly, during early childhood and, secondly during the teenage years. Approximately, 90-95% of adults have been infected with EBV and for many this will have been a subclinical event. EBV infection can be associated with significant morbidity and mortality; for example, primary infection in older children or adults is the leading cause of infectious mononucleosis (IM). A disrupted immune response either iatrogenically induced or through genetic defects can result in lymphoproliferative disease. Finally, EBV is oncogenic and is associated with several malignancies. For these reasons, vaccination to prevent the damaging aspects of EBV infection is an attractive intervention. No EBV vaccines have been licensed and the prophylactic vaccine furthest along in clinical trials contains the major virus glycoprotein gp350. In a phase 2 study, the vaccine reduced the rate of IM by 78% but did not prevent EBV infection. An EBV vaccine to prevent IM in adolescence or young adulthood is the most likely population-based vaccine strategy to be tested and adopted. National registry studies will need to be done to track the incidence of MS in EBV-vaccinated and unvaccinated people to see an effect of the vaccine on MS. Assessment of vaccine efficacy with MS being a delayed consequence of EBV infection with the average age of onset being approximately 30 years of age represents multiple challenges.

Effects of substance P on human cerebral microvascular endothelial cell line hCMEC/D3 are mediated exclusively through a truncated NK-1 receptor and depend on cell confluence.

The neuropeptide substance P (SP) mediates pain transmission, immune modulation, vasodilation and neurogenic inflammation. Its role in the peripheral nervous system has been well characterised. However, its actions on the blood-brain barrier (BBB) are less clear and warrant further study. The aim of this study was to characterise the effect of SP on the brain microvascular endothelial cells using the immortalized human brain microvascular endothelial cell line hCMEC/D3. As part of our studies, we have evaluated changes in expression, at mRNA and protein levels, of genes involved in the function of the blood-brain barrier such as occludin, induced by exposure to SP. We show that the effect of SP is dependent on cell confluence status. Thus, at low confluence but not at full confluence, SP treatment reduced occludin expression. The expression of the SP receptor, neurokinin-1 receptor (NK-1R) (the truncated form of the receptor expressed exclusively in this cell line) was also modulated in a similar pattern. SP treatment stimulated extracellular signal-regulated kinase (Erk2) phosphorylation which was not associated to changes in Interleukin-6 (IL-6), Interleukin-8 (IL-8), or Intercellular Adhesion Molecule 1 (ICAM-1) protein expression. In addition, SP treatment effectively recovered nitric oxide production on cells exposed to tumour necrosis factor alpha (TNF-α). SP did not trigger intracellular calcium release in hCMEC/D3 cells. We conclude that hCMEC/D3 cells are partially responsive to SP, that the effects are mediated through the truncated form of the receptor and are dependent on the confluence status of these cells.

Clinical Utility of Cerebrospinal Fluid Aß42 and Tau Measures in Diagnosing Mild Cognitive Impairment in Early Onset Dementia.

BACKGROUND: The differentiation of a preclinical or prodromal Alzheimer's disease (AD) is challenging particularly in patients with early onset Alzheimer's or related dementias (EOARD). We report our experience on diagnostic lumbar puncture to diagnose EOARD at a tertiary neurocognitive referral center in Nottingham, England from March 2018 to October 2020. OBJECTIVE: To assess amyloid-ß42 (Aß42), total tau, and Thr181-phosphorylated tau (p-tau) measurements in the cerebrospinal fluid (CSF) in patients with mild cognitive impairment (MCI) and in relation to their follow-up cognitive performance. METHODS: Thirty participants aged 32-68 years old (mean 59 years; 57% female) were included. Clinical diagnosis was based on clinical presentation, neurocognitive profile, neuroradiological features (MRI, FDG-PET CT) and CSF Aß42, total tau, and p-tau measurements. RESULTS: Patients with MCI who progressed to AD (prodromal AD) had significantly higher CSF total (797.63 pg/ml) and p-tau (82.31 pg/ml), and lower Aß42 levels (398.94 pg/ml) in comparison to their counterparts with stable MCI (total tau 303.67 pg/ml, p-tau 43.56 pg/ml, Aß42 873.44 pg/ml) (p < 0.01 for CSF total and p-tau measures and p < 0.0001 for CSF Aß42 measures). None of the CSF biomarkers correlated with any of the cognitive performance measures. Principal component analysis confirmed that the clinical diagnosis of MCI secondary to AD, namely prodromal AD (as per NIA-AA criteria) in younger adults, was associated with decreased CSF Aß42. CONCLUSION: In early onset AD, low levels of CSF Aß42 appear to be more sensitive than total and p-tau measures in differentiating AD MCI from other forms of dementia. Further work on larger samples of EOARD in clinical practice will address the cost effectiveness of making an earlier diagnosis.

PAF-R on activated T cells: Role in the IL-23/Th17 pathway and relevance to multiple sclerosis.

IL-23 is a potent stimulus for Th17 cells. These cells have a distinct developmental pathway from Th1 cells induced by IL-12 and are implicated in autoimmune and inflammatory disorders including multiple sclerosis (MS). TGF-ß, IL-6, and IL-1, the transcriptional regulator RORγt (RORC) and IL-23 are implicated in Th17 development and maintenance. In human polyclonally activated T cells, IL-23 enhances IL-17 production. The aims of our study were: 1). To validate microarray results showing preferential expression of platelet activating factor receptor (PAF-R) on IL-23 stimulated T cells. 2). To determine whether PAF-R on activated T cells is functional, whether it is co-regulated with Th17-associated molecules, and whether it is implicated in Th17 function. 3). To determine PAF-R expression in MS. We show that PAF-R is expressed on activated T cells, and is inducible by IL-23 and IL-17, which in turn are induced by PAF binding to PAF-R. PAF-R is co-expressed with IL-17 and regulated similarly with Th17 markers IL-17A, IL-17F, IL-22 and RORC. PAF-R is upregulated on PBMC and T cells of MS patients, and levels correlate with IL-17 and with MS disability scores. Our results show that PAF-R on T cells is associated with the Th17 phenotype and function. Clinical Implications Targeting PAF-R may interfere with Th17 function and offer therapeutic intervention in Th17-associated conditions, including MS.

Subcutaneous cladribine to treat multiple sclerosis: experience in 208 patients.

OBJECTIVE: To report on safety and effectiveness of subcutaneous cladribine (Litak®) in multiple sclerosis (MS) patients. METHODS: Litak® was offered to MS-patients irrespective of disease course. Litak® 10 mg was administered for 3-4 days during week 1. Based on lymphocyte count at week 4, patients received another 0-3 doses at week 5. A second course was administered 11 months later. Follow-up included adverse events, relapses, expanded disability status scale (EDSS), 9-hole-peg and Timed-25-foot-walking tests, no-evidence-of-disease-activity (NEDA), no-evidence-of-progression-or-active-disease (NEPAD), MRI, cerebrospinal fluid (CSF) neurofilament light chain (NfL), and lymphocyte counts. RESULTS: In all, 208 patients received at least one course of treatment. Age at baseline was 44 (17-72) years and EDSS 0-8.5. Cladribine was generally well tolerated. One myocardial infarction, one breast cancer, and three severe skin reactions occurred without long-term sequelae. Two patients died (one pneumonia, one encephalitis). Lymphopenia grade 3 occurred in 5% and grade 4 in 0.5%. In 94 out of 116 pwMS with baseline and follow-up (BaFU) data after two treatment courses, EDSS remained stable or improved. At 18 months, 64% of patients with relapsing MS and BaFU data (n = 39) had NEDA. At 19 months, 62% of patients with progressive MS and BaFU data (n = 13) had NEPAD. Of n = 13 patients whose CSF-NfL at baseline was elevated, 77% were normalised within 12 months. CONCLUSIONS: Litak® was well tolerated. Effectiveness in relapsing MS appeared similar to cladribine tablets and was encouraging in progressive MS. Our data suggest cladribine may be safe and effective in MS-patients irrespective of their disease stage.

An Unusual Cause of Cranial Dural Thickening.

We describe an unusual cause of cranial dural thickening in an elderly female with a chronic meningeal inflammatory process. A 70-year-old ethnically Chinese, Singaporean female presented with a history of chronic daily headache with no other meningeal signs. Serial MRI brains showed progressive pachymeningeal and leptomeningeal enhancement in the left frontal region with underlying vasogenic oedema, similar appearances in the right frontal region to a lesser extent, and persistent inflammatory changes in her bilateral paranasal sinuses. Investigative work-up showed a chronically raised ESR with a normal CRP, negative ANCA, and a chronically raised serum IgA kappa paraprotein. Bone marrow trephine biopsy was suggestive of a low level plasma cell disorder. Olfactory cleft biopsy showed no evidence of IgG4-related disease or vasculitis and no significant plasma cell infiltrate. Histopathological examination from a meningeal biopsy revealed a diagnosis of an en-plaque meningioma (the WHO, 2016; Grade I) causing an unusual granulomatous reaction. We discuss the radiological and histological relations of this rare form of meningioma. Clinicians can consider en-plaque meningioma in the differential diagnosis of linear dural thickening and enhancement.

CCN3 is dynamically regulated by treatment and disease state in multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is an immune-mediated disease that damages myelin in the central nervous system (CNS). We investigated the profile of CCN3, a known regulator of immune function and a potential mediator of myelin regeneration, in multiple sclerosis in the context of disease state and disease-modifying treatment. METHODS: CCN3 expression was analysed in plasma, immune cells, CSF and brain tissue of MS patient groups and control subjects by ELISA, western blot, qPCR, histology and in situ hybridization. RESULTS: Plasma CCN3 levels were comparable between collective MS cohorts and controls but were significantly higher in progressive versus relapsing-remitting MS and between patients on interferon-ß versus natalizumab. Higher body mass index was associated with higher CCN3 levels in controls as reported previously, but this correlation was absent in MS patients. A significant positive correlation was found between CCN3 levels in matched plasma and CSF of MS patients which was absent in a comparator group of idiopathic intracranial hypertension patients. PBMCs and CD4+ T cells significantly upregulated CCN3 mRNA in MS patients versus controls. In the CNS, CCN3 was detected in neurons, astrocytes and blood vessels. Although overall levels of area immunoreactivity were comparable between non-affected, demyelinated and remyelinated tissue, the profile of expression varied dramatically. CONCLUSIONS: This investigation provides the first comprehensive profile of CCN3 expression in MS and provides rationale to determine if CCN3 contributes to neuroimmunological functions in the CNS.

An Absence of Epstein-Barr Virus Reactivation and Associations with Disease Activity in People with Multiple Sclerosis Undergoing Therapeutic Hookworm Vaccination.

Background: Epstein-Barr virus (EBV) infection is strongly associated with multiple sclerosis (MS). Helminth infection can downregulate antiviral immune responses, potentially protecting against MS, but with a theoretical risk for reactivating latent EBV infection. Objective: To investigate parameters of EBV infection and their relationship with disease activity in people with MS (PwMS) therapeutically vaccinated with Necator americanus (hookworm). Methods: Sequential serum samples from 51 PwMS; 26 therapeutically infected (25 larvae) with N. americanus and 25 controls were tested for EBV virus capsid antigen (VCA) IgG and IgM, EBV nuclear antigen-1 (EBNA-1) IgG, and EBV early antigen (EA) IgG. Disease activity was assessed by periodic MRI. Significance was set at p < 0.05. Results: All PwMS were EBV VCA IgG and EBNA-1 IgG positive, and 35.2% were EBV EA IgG positive. EBV antibody levels were generally stable, and EBV reactivation in PwMS was not demonstrated by significant increases in IgG titre over 12 months. Disease activity was most frequent in PwMS possessing high levels of EBV VCA IgG (>600 units/mL) or EBNA-1 IgG (>150 units/mL); however, there was no association with hookworm treatment. Interpretation: Therapeutic hookworm vaccination was not associated with EBV reactivation. Multiple sclerosis disease activity was associated with high levels of EBV VCA IgG or EBNA-1 IgG.

Increased IL-2 and Reduced TGF-ß Upon T-Cell Stimulation are Associated with GM-CSF Upregulation in Multiple Immune Cell Types in Multiple Sclerosis.

Granulocyte macrophage colony stimulating factor (GM-CSF) is a pro-inflammatory cytokine produced by immune cells. Recent evidence suggests that GM-CSF plays an important role in multiple sclerosis (MS) pathogenesis. We investigated the expression and regulation of GM-CSF in different immune cells in MS. We also investigated the differentiation and frequency of GM-CSF-producing Th cells that do not co-express interferon (IFN)-γ or interleukin-17 (IL-17) (Th-GM cells) in MS. We found a significant increase in the percentage of GM-CSF-expressing Th cells, Th1 cells, Th-GM cells, cytotoxic T (Tc) cells, monocytes, natural killer (NK) cells, and B cells in PBMC from MS patients stimulated with T cell stimuli. Stimulated PBMC culture supernatants from MS patients contained significantly higher levels of IL-2, IL-12, IL-1ß, and GM-CSF and significantly lower levels of transforming growth factor (TGF-)ß. Blocking IL-2 reduced the frequency of Th-GM cells in PBMC from MS patients. The frequency of Th-GM cells differentiated in vitro from naïve CD4+ T cells was significantly higher in MS patients and was further increased in MS with IL-2 stimulation. These findings suggest that all main immune cell subsets produce more GM-CSF in MS after in vitro stimulation, which is associated with defective TGF-ß and increased IL-2 and IL-12 production. Th-GM cells are increased in MS. GM-CSF may be a potential therapeutic target in MS.

Hookworm Treatment for Relapsing Multiple Sclerosis: A Randomized Double-Blinded Placebo-Controlled Trial.

Importance: Studies suggest gut worms induce immune responses that can protect against multiple sclerosis (MS). To our knowledge, there are no controlled treatment trials with helminth in MS. Objective: To determine whether hookworm treatment has effects on magnetic resonance imaging (MRI) activity and T regulatory cells in relapsing MS. Design, Setting, and Participants: This 9-month double-blind, randomized, placebo-controlled trial was conducted between September 2012 and March 2016 in a modified intention-to-treat population (the data were analyzed June 2018) at the University of Nottingham, Queen's Medical Centre, a single tertiary referral center. Patients aged 18 to 61 years with relapsing MS without disease-modifying treatment were recruited from the MS clinic. Seventy-three patients were screened; of these, 71 were recruited (2 ineligible/declined). Interventions: Patients were randomized (1:1) to receive either 25 Necator americanus larvae transcutaneously or placebo. The MRI scans were performed monthly during months 3 to 9 and 3 months posttreatment. Main Outcomes and Measures: The primary end point was the cumulative number of new/enlarging T2/new enhancing T1 lesions at month 9. The secondary end point was the percentage of cluster of differentiation (CD) 4+CD25highCD127negT regulatory cells in peripheral blood. Results: Patients (mean [SD] age, 45 [9.5] years; 50 women [71%]) were randomized to receive hookworm (35 [49.3%]) or placebo (36 [50.7%]). Sixty-six patients (93.0%) completed the trial. The median cumulative numbers of new/enlarging/enhancing lesions were not significantly different between the groups by preplanned Mann-Whitney U tests, which lose power with tied data (high number of zeroactivity MRIs in the hookworm group, 18/35 [51.4%] vs 10/36 [27.8%] in the placebo group). The percentage of CD4+CD25highCD127negT cells increased at month 9 in the hookworm group (hookworm, 32 [4.4%]; placebo, 34 [3.9%]; P = .01). No patients withdrew because of adverse effects. There were no differences in adverse events between groups except more application-site skin discomfort in the hookworm group (82% vs 28%). There were 5 relapses (14.3%) in the hookworm group vs 11 (30.6%) receiving placebo. Conclusions and Relevance: Treatment with hookworm was safe and well tolerated. The primary outcome did not reach significance, likely because of a low level of disease activity. Hookworm infection increased T regulatory cells, suggesting an immunobiological effect of hookworm. It appears that a living organism can precipitate immunoregulatory changes that may affect MS disease activity. Trial Registration: ClinicalTrials.gov Identifier: NCT01470521.