Deciphering the shift from benign to active relapsing-remitting multiple sclerosis: Insights into T regulatory cell dysfunction and apoptosis regulation.

BACKGROUND: Relapsing-remitting multiple sclerosis (RRMS), a common demyelinating disease among young adults, follows a benign course in 10-15% of cases, where patients experience minimal neurological disability for a decade following disease onset. However, there is potential for these benign cases to transition into a clinically active, relapsing state. OBJECTIVE: To elucidate the biological mechanisms underlying the transition from benign to active RRMS using gene expression analysis. METHODS: We employed complementary-DNA microarrays to examine peripheral-blood gene expression patterns in patients with benign MS, defined as having a disease duration exceeding 10 years and an Expanded Disability Status Scale (EDSS) score of ≤3.0. We compared the gene expression pattern between patients who switched to active disease (Switching BMS) with those who maintained a benign state (Permanent-BMS) during an additional 5-year follow-up. RESULTS: We identified two primary mechanisms linked to the transition from benign MS to clinically active disease. The first involves the suppression of regulatory T cell activity, and the second pertains to the dysfunction of nuclear receptor 4 A family-dependent apoptosis. These mechanisms collectively contribute to an augmented autoimmune response and increased disease activity. CONCLUSIONS: The intricate gene regulatory networks that operate in switching-BMS are related to suppression of immune tolerance and aberrant apoptosis. These findings may lead to new therapeutic targets to prevent the escalation to active disease.

In-depth characterization of long-term humoral and cellular immune responses to COVID-19m-RNA vaccination in multiple sclerosis patients treated with teriflunomide or alemtuzumab.

BACKGROUND: The impact of disease-modifying therapies on the efficacy to mount appropriate immune responses to COVID-19 vaccination in patients with multiple sclerosis (MS) is currently under investigation. OBJECTIVE: To characterize long-term humoral and cellular immunity in mRNA-COVID-19 MS vaccinees treated with teriflunomide or alemtuzumab. METHODS: We prospectively measured SARS-COV-2 IgG, memory B-cells specific for SARS-CoV-2 RBD, and memory T-cells secreting IFN-γ and/or IL-2, in MS patients vaccinated with BNT162b2-COVID-19 vaccine before, 1, 3 and 6 months after the second vaccine dose, and 3-6 months following vaccine booster. RESULTS: Patients were either untreated (N = 31, 21 females), under treatment with teriflunomide (N = 30, 23 females, median treatment duration 3.7 years, range 1.5-7.0 years), or under treatment with alemtuzumab (N = 12, 9 females, median time from last dosing 15.9 months, range 1.8-28.7 months). None of the patients had clinical SARS-CoV-2 or immune evidence for prior infection. Spike IgG titers were similar between untreated, teriflunomide and alemtuzumab treated MS patients both at 1 month (median 1320.7, 25-75 IQR 850.9-3152.8 vs. median 901.7, 25-75 IQR 618.5-1495.8, vs. median 1291.9, 25-75 IQR 590.8-2950.9, BAU/ml, respectively), at 3 months (median 1388.8, 25-75 1064.6-2347.6 vs. median 1164.3 25-75 IQR 726.4-1399.6, vs. median 837.2, 25-75 IQR 739.4-1868.5 BAU/ml, respectively), and at 6 months (median 437.0, 25-75 206.1-1161.3 vs. median 494.3, 25-75 IQR 214.6-716.5, vs. median 176.3, 25-75 IQR 72.3-328.8 BAU/ml, respectively) after the second vaccine dose. Specific SARS-CoV-2 memory B cells were detected in 41.9%, 40.0% and 41.7% of subjects at 1 month, in 32.3%, 43.3% and 25% at 3 months, and in 32.3%, 40.0%, 33.3% at 6 months following vaccination in untreated, teriflunomide treated and alemtuzumab treated MS patients, respectively. Specific SARS-CoV-2 memory T cells were found in 48.4%, 46.7% and 41.7 at 1 month, in 41.9%, 56.7% and 41.7% at 3 months, and in 38.7%, 50.0%, and 41.7% at 6 months, of untreated, teriflunomide-treated and alemtuzumab -treated MS patients, respectively. Administration of a third vaccine booster significantly increased both humoral and cellular responses in all patients. CONCLUSIONS: MS patients treated with teriflunomide or alemtuzumab achieved effective humoral and cellular immune responses up to 6 months following second COVID-19 vaccination. Immune responses were reinforced following the third vaccine booster.

Faster progression to multiple sclerosis disability is linked to neuronal pathways associated with neurodegeneration: An ethnicity study.

Although the causes of multiple sclerosis are largely unknown, genetic and environmental components play an important role. Geographic distribution, varying with latitude, reflects both genetic and environmental influences. We conducted a retrospective exploratory observational study to characterize the disability progression of 2396 Jewish patients with relapsing-remitting multiple sclerosis, followed at the Sheba Multiple Sclerosis Center, Tel-Aviv, Israel; 188 patients who originated in Iraq and 2207 patients who originated in northern Europe. Peripheral blood microarray gene expression analysis was performed in a subgroup of patients to identify molecular pathways associated with faster disability progression. During a follow-up period of 18.8 and 19.8 years, respectively, 51.6% of patients with an Iraqi origin progressed to moderate disability defined as expanded disability status scale (EDSS) score of 3.0 to 5.5, compared to 44.2% of patients with a northern European origin (odds ratio 1.347, 95% CI 1.0-1.815, p = 0.049). An Iraqi origin was associated with increased risk of progression to moderate disability adjusted for sex, disease duration, age at onset, and treatment with immunomodulatory drugs (hazard ratio 1.323; 95% CI, 1.049-1.668, p = 0.02), but not to severe disability defined as EDSS score > = 6.0 (i.e., walking aids are required for a distance of 100 meters, (hazard ratio 1.311; 95% CI, 0.918-1.874, p = 0.136). Gene expression analysis disclosed 98 differentially expressed genes (79 over-expressed and 19 under-expressed) between relapsing-remitting multiple sclerosis patients of Iraqi origin (N = 17) and northern European (N = 34) origin. Interestingly, this gene expression was enriched with genes related to neuronal pathways associated with morphology of axons, branching of neurites, proliferation of neocortical neurons, and formation of myelin sheath, suggesting an augmented process of neurodegeneration in relapsing-remitting multiple sclerosis patients with an Iraqi origin. The study results suggest that relapsing-remitting multiple sclerosis patients with an Iraqi origin progress faster to disability possibly due to an enhanced process of neurodegeneration.

COVID-19 Alpha Variant (B.1.1.7): Humoral, memory B and T cells in COVID-19 pediatric convalescents.

BACKGROUND: Studies of anti-SARS-CoV-2 humoral and adaptive response in COVID-19 non-vaccinated pediatric convalescents are controversial and further evidence from the pediatric population are needed. OBJECTIVES: To elucidate SARS-CoV-2 humoral and memory B- and T-cells responses in pediatric convalescents as compared with the adult. METHODS: Blood samples were obtained from 80 non-vaccinated, IgG-positive, COVID-19 convalescents (age 8.0-61.0 years), 4.0 months from onset. Frequency of responders and magnitudes of SARS-COV-2 IgG, memory B-cells (MBC) and IFNg- and IL2-secreting memory T-cells (MTC) in response to immuno-dominant peptide pools in pediatric, young adults and middle-aged adults with onset age 8-18 years (N = 20), 19-39 years (N = 30) and 40-61 years (N = 30), respectively, were analyzed. SARS-CoV-2 IgG were detected by ELISA (Euroimmun, Germany). MBC, IFNg-, IL2- and IFNg+IL2-secreting MTC (IFNg-MTC, IL2-MTC and IFNg+IL2-MTC) were detected using FluoroSpot (Mabtech, Sweden). RESULTS: MBC level was lower in pediatric as compared with the middle-aged adults (median 12.75 interquartile range [IQR] 4.27-33.7 and 32.0 IQR 6.0-124.2, respectively, p = .003). MBC level in young adults was lower than in middle-aged adults (median 18.5 IQR 1.7-43.8 and 32.0 IQR 6.0-124.2, respectively, p = .006). The level of IL2-MTC was lower in the pediatric group as compared with middle aged-adults (median 2.1 IQR 0-16.9 and 28.6 IQR 11-49.6, respectively, p < .03) and in young adults lower than in middle-aged adults (median 1.45 IQR 0-18.6 and 28.6 IQR 11-49.6, respectively, p = .02). In addition, the level of IFNg-MTC was lower in pediatric as compared with young adults (median 4.25 IQR 0.0-15.0 and 20.9 IQR 0-75.2, respectively, p = .05). The level of IgG was comparable between pediatric and both young and middle-aged adult groups (4.82 ± 2.95, 3.70 ± 2.65 and 4.9 ± 2.94, respectively, p > .34). CONCLUSION: Non-vaccinated COVID-19 pediatric convalescents have lower adaptive immune responses than adults sustaining the recommendation for vaccination of the pediatric population.

Immune response to the third COVID-19 vaccine dose is related to lymphocyte count in multiple sclerosis patients treated with fingolimod.

BACKGROUND: The majority of multiple sclerosis [MS] patients treated with fingolimod fail to develop a protective level of IgG humoral and adaptive cellular immune responses following full BNT162b2 SARS-CoV-2 vaccination. OBJECTIVE: To compare the efficacy of the third COVID-19 vaccine dose in vaccine non-responders fingolimod-treated MS patients. STUDY DESIGN: This is a prospective 3-month, single-center, randomized clinical trial. METHODS: Twenty relapsing MS patients who had been on fingolimod therapy ≥ 12 months and failed to develop humoral IgG immune response to 2-dose Pfizer BNT162b2 COVID-19 vaccination were randomized into two groups: fingolimod-continuation group and fingolimod-discontinuation group. Humoral and memory cellular immune responses were assessed within 1 and 3 months following the third Pfizer BNT162b2 vaccine dose and compared between the groups. RESULTS: A higher rate of patients in the fingolimod-discontinuation group [n = 8/10] compared to fingolimod-continuation group [n = 2/10] developed positive SARS-COV-2 IgG. Median IgG titer 1 month following the third dose was 202.3 BAU/ml vs. 26.4 BAU/ml, respectively, p = 0.022. The development of IgG humoral response correlated with absolute lymphocyte count. Specific SARS-COV-2 memory B cell and T cell immune responses were not detected in both groups, either at 1 month or 3 months following the third COVID-19 vaccine dose. CONCLUSIONS: Short period of fingolimod treatment discontinuation was associated with the development of humoral protection but not with adaptive cellular immunity.

COVID-19 vaccination in patients with multiple sclerosis: Safety and humoral efficacy of the third booster dose.

BACKGROUND: As immunity against SARS-COV-2 wanes following first and second doses of vaccination, a third dose is administered in several countries around the world. Similarly to the first doses, risks related to vaccination and humoral immune response in patients with multiple sclerosis (MS) need to be assessed. OBJECTIVE: Characterize safety and humoral immune response following the third dose of COVID-19 vaccination in a large cohort of MS patients. METHODS: We assessed the safety of the third dose of the BNT162b2-COVID-19 mRNA vaccination in adult MS patients and evaluated SARS-CoV-2 IgG response. RESULTS: Two hundred and eleven adult MS patients received a third dose of BNT162b2 COVID-19 vaccination. Median follow up time was 66 days from vaccine administration (IQR 54-84). The frequency of any adverse event was 54.5%, with the most common reported adverse events being fatigue, local pain at the injection site, fever and muscle or joint pain. Transient increase in MS symptoms was reported in 3.8% of patients, none of them requiring treatment. The rate of acute relapses treated with IV steroids was 3.3%. In a sub-group of 55 patients, 20 untreated and 35 treated with vaccination-safe disease-modifying treatments, SARS-CoV-2 IgG levels increased 21-fold (median ± SD 21.6 ± 53.05). CONCLUSIONS: The third dose of COVID-19-BNT162b2 vaccine proved safe for MS patients, with no increased risk of relapse activity. Untreated patients and patients treated with vaccination-safe disease-modifying treatments show significant increase in SARS-CoV-2 IgG levels following the third dose of vaccination.

SARS-CoV-2 memory B and T cell profiles in mild COVID-19 convalescent patients.

OBJECTIVES: Antiviral adaptive immunity involves memory B cells (MBC) and memory T cells (MTC). The dynamics of MBC and MTC in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) convalescents warrant further investigation. METHODS: In this cross-sectional and longitudinal study, blood-derived MBC and MTC responses were evaluated in 68 anti-spike IgG-positive mild coronavirus disease 2019 (COVID-19) convalescents at visit 1, between 1 and 7 months (median 4.1 months) after disease onset. SARS-CoV-2 anti-spike IgG was determined by ELISA, MBC by SARS-CoV-2-specific receptor binding domain (RBD) ELISpot, and interferon gamma (IFN-γ)-, interleukin 2 (IL2)-, and IFN-γ+IL2-secreting MTC by IFN-γ and IL2 SARS-CoV-2 FluoroSpot. For 24 patients sampled at the first visit, the IgG, MBC, and MTC analyses were also performed 3 months later at the second visit. RESULTS: Seventy-two percent of convalescents were both MBC- and MTC-positive, 18% were MBC-positive and MTC-negative, and 10% were MTC-positive and MBC-negative. The peak MBC response level was detected at 3 months after COVID-19 onset and persisted up to 7 months post infection. Significant MTC levels were detected 1 month after onset in response to S1, S2_N, and SNMO peptide pools. The frequency and magnitude of the MTC response to SNMO was higher than those to S1 and S2_N. Longitudinal analysis demonstrated that even when specific humoral immunity declined, the cellular immunity persisted. CONCLUSIONS: The study findings demonstrate the durability of adaptive cellular immunity at least for 7 months after SARS-CoV-2 infection, suggesting long-lasting protection.

Humoral immune response in multiple sclerosis patients following PfizerBNT162b2 COVID19 vaccination: Up to 6 months cross-sectional study.

Appropriate immune response following COVID-19 vaccination is important in the context of disease-modifying treatments (DMTs). In a prospective cross-sectional study, we determined SARS-COV-2 IgG response up to 6 months following PfizerBNT162b2 vaccination in 414 multiple sclerosis (MS) patients and 89 healthy subjects. Protective response was demonstrated in untreated MS patients (N = 76, 100%), treated with Cladribine (N = 48, 100%), Dimethyl fumarate (N = 35, 100%), Natalizumab (N = 32, 100%), and Teriflunomide (N = 39, 100%), similarly to healthy subjects (N = 89, 97.8%). Response was decreased in Fingolimod (N = 42, 9.5%), Ocrelizumab (N = 114, 22.8%) and Alemtuzumab (N = 22, 86.4%) treated patients. IgG response can help tailor adequate vaccine guidelines for MS patients under various DMTs.

Humoral immune response to COVID-19 mRNA vaccine in patients with multiple sclerosis treated with high-efficacy disease-modifying therapies.

BACKGROUND AND AIMS: The National Multiple Sclerosis Society and other expert organizations recommended that all patients with multiple sclerosis (MS) should be vaccinated against COVID-19. However, the effect of disease-modifying therapies (DMTs) on the efficacy to mount an appropriate immune response is unknown. We aimed to characterize humoral immunity in mRNA-COVID-19 MS vaccinees treated with high-efficacy DMTs. METHODS: We measured SARS-CoV-2 IgG response using anti-spike protein-based serology (EUROIMMUN) in 125 MS patients vaccinated with BNT162b2-COVID-19 vaccine 1 month after the second dose. Patients were either untreated or under treatment with fingolimod, cladribine, or ocrelizumab. A group of healthy subjects similarly vaccinated served as control. The percent of subjects that developed protective antibodies, the titer, and the time from the last dosing were evaluated. RESULTS: Protective humoral immunity of 97.9%, 100%, 100%, 22.7%, and 3.8%, was observed in COVID-19 vaccinated healthy subjects (N = 47), untreated MS patients (N = 32), and MS patients treated with cladribine (N = 23), ocrelizumab (N = 44), and fingolimod (N = 26), respectively. SARS-CoV-2 IgG antibody titer was high in healthy subjects, untreated MS patients, and MS patients under cladribine treatment, within 29.5-55 days after the second vaccine dose. Only 22.7% of patients treated with ocrelizumab developed humoral IgG response irrespective to normal absolute lymphocyte count. Most fingolimod-treated MS patients had very low lymphocyte count and failed to develop SARS-COV-2 antibodies. Age, disease duration, and time from the last dosing did not affect humoral response to COVID-19 vaccination. CONCLUSIONS: Cladribine treatment does not impair humoral response to COVID-19 vaccination. We recommend postponing ocrelizumab treatment in MS patients willing to be vaccinated as a protective humoral response can be expected only in some. We do not recommend vaccinating MS patients treated with fingolimod as a protective humoral response is not expected.

SARS-CoV-2 antibody dynamics and B-cell memory response over time in COVID-19 convalescent subjects.

OBJECTIVES: The worldwide spread of coronavirus disease 2019 (COVID-19) highlights the need for assessment of long-term humoral immunity in convalescent subjects. Our objectives were to evaluate long-term IgG antibody response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and B-cell memory response in COVID-19 convalescent subjects. METHODS: Blood samples were collected from a cohort of subjects recovering from COVID-19 and from healthy subjects who donated blood. SARS-CoV-2 IgG antibodies were quantitatively detected by ELISA using anti-S1 spike IgG. SARS-CoV-2 spike-specific IgG memory B cells were evaluated by reversed B-cell FluroSpot based on human IgG SARS-CoV-2 receptor-binding domain in a randomly selected group of subjects recovering from COVID-19. Statistical analysis was performed with clinical variables and time post COVID-19 infection. RESULTS: Antibody response was not detected in 26 of 392 COVID-19 convalescent subjects (6.6%). Over a period of 9 months, the level of antibodies decreased by 50% but stabilized at 6 months, and a protective level prevailed for up to 9 months. No differences were found regarding IgG SARS-CoV-2 antibody levels for age, gender, and major blood types over time. Over time, asymptomatic COVID-19 subjects did not differ in antibody level from subjects with mild to severe disease. Repeated paired IgG SARS-CoV-2 antibody level analyses disclosed that, over 6 and 9 months, 15.3% (nine of 59) and 15.8% (three of 19) of subjects became SARS-CoV-2 IgG-seronegative, respectively, all with a low antibody level at 3 months. Rate of antibody decline was not affected by age, gender, or clinical symptomatology. In a subgroup of recovering subjects, memory B-cell response up to 9 months post-COVID-19 infection was undetectable in 31.8% of subjects (14/44), and there was no correlation with age, SARS-CoV-2 antibody level, or time post infection. CONCLUSIONS: The majority of convalescent COVID-19 subjects develop an IgG SARS-CoV-2 antibody response and a protective level prevails over a period of up to 9 months, regardless of age, gender, major blood types or clinical symptomatology.

COVID-19 vaccination in patients with multiple sclerosis: What we have learnt by February 2021.

BACKGROUND: Since vaccination against coronavirus disease 2019 (COVID-19) became available, risks related to vaccinating patients with multiple sclerosis (MS) need to be carefully assessed. OBJECTIVE: Characterize safety and occurrence of immediate relapses following COVID-19 vaccination in a large cohort of MS patients. METHODS: We assessed the safety of BNT162b2 COVID-19 vaccination in adult MS patients. RESULTS: Between 20 December 2020 and 25 January 2021, 555 MS patients received the first dose of BNT162b2 vaccine and 435 received the second dose. There were three cases of COVID-19 infection encountered after the first dose. Safety profile of COVID-19 vaccine was characterized by pain at the injection site, fatigue, and headache. No increased risk of relapse activity was noted over a median follow-up of 20 and 38 days after first and second vaccine doses, respectively. The rate of patients with acute relapse was 2.1% and 1.6% following the first and second doses, respectively, similar to the rate in non-vaccinating patients during the corresponding period. Mild increase in the rate of adverse events was noted in younger patients (18-55 years), among patients with lower disability (Expanded Disability Status Scale (EDSS) ⩽3.0), and in patients treated with immunomodulatory drugs. CONCLUSION: COVID-19 BNT162b2 vaccine proved safe for MS patients. No increased risk of relapse activity was noted.

Clinical and transcriptional recovery profiles in pediatric and adult multiple sclerosis patients.

OBJECTIVE: To determine whether pediatric-onset multiple sclerosis (POMS) and adults-onset multiple sclerosis (AOMS) patients are different in initial disease severity and recovery and to investigate the associations with peripheral blood mononuclear cells (PBMCs) transcriptional profiles. METHODS: Clinical and radiological severity of first and second relapses and 6-month recovery were analyzed in 2153 multiple sclerosis (MS) patients and compared between POMS (onset at 8-18years old) and AOMS (onset at 19-40 years old) patients. PBMCs transcriptomes of 15 POMS and 15 gender-matched AOMS patients were analyzed 6 months after the first relapse and compared to 55 age-matched healthy controls. Differentially Expressed Genes (DEGs) with a false discovery rate ≤ 10% were evaluated using the Partek software. RESULTS: POMS had increased Expanded Disability Status Scale (EDSS) score at first and second relapses, higher brain gadolinium-enhancing T1-lesions volume at first relapse, and more complete recovery after both relapses compared to AOMS. POMS patients, who recovered completely from the first relapse, were characterized by 19 DEGs that were mainly related to suppression of antigen presentation. Six upstream regulators of these genes were differentially expressed between pediatric and adult healthy controls. POMS patients, who showed no recovery from the first relapse, were characterized by 28 DEGs that were mainly associated with B-cell activation. Five upstream regulators of these genes were differentially expressed between pediatric and adult healthy controls. INTERPRETATION: POMS patients may have more severe first and second relapses than AOMS. However, most often, POMS have better recovery that may be attributed to PBMCs age-related transcriptional profiles associated with antigen presentation and B-cell activation.

RAM-589.555 favors neuroprotective and anti-inflammatory profile of CNS-resident glial cells in acute relapse EAE affected mice.

BACKGROUND: Targeting RNA polymerase-1 (POL1) machinery is a new strategy for suppression of multiple sclerosis (MS) relapse activity. Oral administration of POL1 inhibitor RAM-589.555, which is characterized by high permeability and bioavailability in naïve mice, ameliorates proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE) by suppressing activated autoreactive lymphocytes. We assessed the accessibility of RAM-589.555 to the central nervous system (CNS) of EAE-mice and further investigated its immunomodulatory effects on CNS-resident astro- and micro-glial cells in-vitro and in-vivo. METHODS: Effects of RAM-589.555 on activated microglia and astrocyte viability, proliferation, and secretion of neurotrophic factors were assessed in-vitro. The pharmacokinetic of RAM-589.555 was evaluated in the blood and central nervous system (CNS) of EAE-affected mice. High-dimensional single-cell mass cytometry was applied to characterize the effect of RAM-589.555 on EAE-affected mice's CNS-resident micro- and astroglial cells and CNS-infiltrating immune cells, which were obtained seven days after RAM-589.555 administration at EAE onset. Simultaneously, the expression level of pre-rRNA, the POL1 end product, was assessed in blood cells, microglia, and astrocytes to monitor RAM-589.555 effects. RESULTS: RAM-589.555 demonstrated blood and CNS permeability in EAE mice. In-vitro, incubation with 400 nM of RAM-589.555 significantly reduced viability and proliferation of lipopolysaccharide (LPS)-activated microglia by 70% and 45% (p < 0.05), respectively, while tumor necrosis factor α (TNFα)-activated astrocytes were not affected. The secretion of neurotrophic factors was preserved. Furthermore, 7 days after administration of RAM-589.555 at EAE onset, the level of pre-rRNA transcript in peripheral blood mononuclear cells (PBMC) was decreased by 38.6% (p = 0.02), while levels of pre-rRNA transcript in microglia and astrocytes remained unchanged. The high-dimensional single-cell mass cytometry analysis showed decreased percentages of CNS-resident microglia and astrocytes, diminished pro-inflammatory cytokines (IL-1ß, IL-6, IL-12, IL-17, TNFα, and IFNγ), and an increase of their anti-inflammatory cytokines (IL-4, IL-10, and TGFß) in RAM-589.555-treated compared to vehicle-treated mice (p < 0.05). CONCLUSIONS: These data correlate RAM-589.555-induced clinical amelioration and its CNS-permeability to decreased CNS-inflammation, and decreased micro- and astrogliosis, while restoring micro- and astroglial anti-inflammatory and neuroprotective capacity.

The Median Eminence, A New Oligodendrogenic Niche in the Adult Mouse Brain.

The subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus in the hippocampus are known as neurogenic niches. We show that the median eminence (ME) of the hypothalamus comprises BrdU+ newly proliferating cells co-expressing NG2 (oligodendrocyte progenitors) and RIP (pre-myelinating oligodendrocytes), suggesting their differentiation toward mature oligodendrocytes (OLs). ME cells can generate neurospheres (NS) in vitro, which differentiate mostly to OLs compared with SVZ-NS that typically generate neurons. Interestingly, this population of oligodendrocyte progenitors is increased in the ME from experimental autoimmune encephalomyelitis (EAE)-affected mice. Notably, the thrombospondin 1 (TSP1) expressed by astrocytes, acts as negative regulator of oligodendrogenesis in vitro and is downregulated in the ME of EAE mice. Importantly, transplanted ME-NS preferentially differentiate to MBP+ OLs compared with SVZ-NS in Shiverer mice. Hence, discovering the ME as a new site for myelin-producing cells has a great importance for advising future therapy for demyelinating diseases and spinal cord injury.

Polymerase-1 pathway activation in acute multiple sclerosis relapse.

BACKGROUND: Increased expression of RNA polymerase 1 (POL1) molecular pathway was reported to be associated with increased disease activity in patients with multiple sclerosis (MS). However, the operating molecular mechanisms that characterize the pattern of acute MS relapse activity has not been thoroughly studied. OBJECTIVE: To assess POL1 pathway expression during acute MS relapse. METHODS: We studied POL1 pathway associated biomarkers during the first acute optic neuritis attack of MS, and in relapsing-remitting MS patients treated with disease-modifying drugs (DMDs) experiencing an acute MS relapse or a radiological relapse using gene expression microarrays and quantitative RT-PCR. RESULTS: In MS patients (N = 6) during the first acute optic neuritis attack POL1 pathway activation was evident by over-expression of POL1 related network including transcription factor UBTF and downstream components of Assembly of RNA POL1 complex (p=1.92E-03). POL1 related biomarkers RRN3, POLR1D and LRPPRC were over-expressed x1.6 (p = .002), ×1.7 (p = .01) and x2.0 (p = .001) times higher respectively, in MS patients (N = 30) during acute clinical relapse as compared with remission. Similarly, in MS patients (N = 21) that presented with a radiological relapse, we observed significant activation of POL1 related biomarkers including RRN3 (p = .01), POLR1D (p = .002), POLR1E (p = .0001) and LRPPRC (p = .006), as compared with remission, as well as overexpression of a large group of genes encoding ribosomal proteins like RPS6KA3 (p = 7.2E-6), RRP8 (p = .0002) and RPCS9 (p = .0008). CONCLUSIONS: Our findings demonstrated increased POL1 pathway activity in acute MS relapse and suggest that targeted inactivation of POL1 pathway represent a novel strategy for a better treatment of acute MS relapse.

Prickle1 as positive regulator of oligodendrocyte differentiation.

Spontaneous neural repair from endogenous neural stem cells' (NSCs) niches occurs in response to central nervous system (CNS) injuries to only a limited extent. Uncovering the mechanisms that control neural repair and can be further manipulated to promote NSCs toward oligodendrocyte progenitors cells (OPCs) and myelinating oligodendrocytes is a major objective. In the current study, we describe high-throughput transcriptional changes in adult mouse subventricular zone (SVZ)-NSCs during differentiation in vitro. In order to identify myelin-specific transcriptional regulators among large transcriptional changes associated with differentiation, we have focused on transcripts encoding transcription factors and regulators showing expression profile that is highly correlated with expression of myelin-encoding genes. We have revealed previously undescribed effect of Prickle1 and Nfe2l3 transcriptional regulators that are positively correlated with expression of myelin basic protein (MBP). Using Prickle1 and Nfe2l3 silencing and immunocytochemistry approaches, we demonstrated that silencing of Prickle1 dramatically decreases differentiation to NG2+OPCs while Nfe2l3 moderately decreases as compared with control siRNA. Moreover, silencing of Prickle1 also decreases maturation of OPCs to MBP+ oligodendrocytes (OLs). Accordingly, overexpression of Prickle1 increases the differentiation of NSCs to CNPase+ pre-myelinating and myelinating MBP+ OLs. In particular, the necessity of Prickle1 for oligodendrocyte differentiation is demonstrated in purified OPCs cultures. Our findings demonstrate the role of Prickle1 in positive regulation of differentiation and maturation of oligodendrocytes suggesting that targeting Prickle1 in CNS injuries and particularly in demyelinating disease could promote generation of myelinating oligodendrocytes from endogenous niches to replenish damaged oligodendrocytes.

Host cell virus entry mechanisms enhance anti-JCV-antibody switch in natalizumab-treated multiple sclerosis patients.

Estimating the individual risk for the development of progressive multifocal leukoencephalopathy (PML) in anti-John Cunningham virus (JCV) antibody-negative patients with multiple sclerosis (MS) treated with natalizumab is a major challenge. A serological conversion occurring under treatment from anti-JCV antibody-negative to positive status may significantly increase this risk. We investigated changes in peripheral blood cells' gene expression induced by natalizumab treatment in anti-JCV antibody-negative MS patients and tested blood transcriptional profile that characterizes patients predisposed to antibody switch under natalizumab treatment. After 3 years of natalizumab treatment, 24.6 % of anti-JCV antibody-negative MS patients switched to become anti-JCV antibody-positive (JCV switchers). Natalizumab induced 946 and 1186 significantly differentiating genes in JCV switchers and non-switchers, respectively. In JCV switchers, the signature was enriched by over-expression of genes associated with the first stages of viral entry to host cells including macropinocytosis (p = 1.82E-06), virus entry via endocytosis (p = 1.60E-06), clathrin-mediated endocytosis (p = 1.13E-04), and caveolar-mediated endocytosis (p = 4.50E-04) pathways. Further analysis to identify pre-existing transcriptional differences that characterize future JCV switchers prior to treatment initiation also demonstrated a transcriptional signature enriched by similar viral entry mechanisms. These findings, verified in an additional independent cohort of natalizumab-treated patients, could lead to future identification of patients that remain anti-JCV antibody-negative thus allowing safe continuation of treatment, as well as the development of future targeted therapeutic interventions to reduce the risk of PML.

Post-CNS-inflammation expression of CXCL12 promotes the endogenous myelin/neuronal repair capacity following spontaneous recovery from multiple sclerosis-like disease.

BACKGROUND: Demyelination and axonal degeneration, hallmarks of multiple sclerosis (MS), are associated with the central nervous system (CNS) inflammation facilitated by C-X-C motif chemokine 12 (CXCL12) chemokine. Both in MS and in experimental autoimmune encephalomyelitis (EAE), the deleterious CNS inflammation has been associated with upregulation of CXCL12 expression in the CNS. We investigated the expression dynamics of CXCL12 in the CNS with progression of clinical EAE and following spontaneous recovery, with a focus on CXCL12 expression in the hippocampal neurogenic dentate gyrus (DG) and in the corpus callosum (CC) of spontaneously recovered mice, and its potential role in promoting the endogenous myelin/neuronal repair capacity. METHODS: CNS tissue sections from mice with different clinical EAE phases or following spontaneous recovery and in vitro differentiated adult neural stem cell cultures were analyzed by immunofluorescent staining and confocal imaging for detecting and enumerating neuronal progenitor cells (NPCs) and oligodendrocyte precursor cells (OPCs) and for expression of CXCL12. RESULTS: Our expression dynamics analysis of CXCL12 in the CNS with EAE progression revealed elevated CXCL12 expression in the DG and CC, which persistently increases following spontaneous recovery even though CNS inflammation has subsided. Correspondingly, the numbers of NPCs and OPCs in the DG and CC, respectively, of EAE-recovered mice increased compared to that of naïve mice (NPCs, p < 0.0001; OPCs, p < 0.00001) or mice with active disease (OPCs, p < 0.0005). Notably, about 30 % of the NPCs and unexpectedly also OPCs (~50 %) express CXCL12, and their numbers in DG and CC, respectively, are higher in EAE-recovered mice compared with naïve mice and also compared with mice with ongoing clinical EAE (CXCL12(+) NPCs, p < 0.005; CXCL12(+) OPCs, p < 0.0005). Moreover, a significant proportion (>20 %) of the CXCL12(+) NPCs and OPCs co-express the CXCL12 receptor, CXCR4, and their numbers significantly increase with recovery from EAE not only relative to naïve mice (p < 0.0002) but also to mice with ongoing EAE (p < 0.004). CONCLUSIONS: These data link CXCL12 expression in the DG and CC of EAE-recovering mice to the promotion of neuro/oligodendrogenesis generating CXCR4(+) CXCL12(+) neuronal and oligodendrocyte progenitor cells endowed with intrinsic neuro/oligondendroglial differentiation potential. These findings highlight the post-CNS-inflammation role of CXCL12 in augmenting the endogenous myelin/neuronal repair capacity in MS-like disease, likely via CXCL12/CXCR4 autocrine signaling.

Transcriptional response to interferon beta-1a treatment in patients with secondary progressive multiple sclerosis.

BACKGROUND: Interferon (IFN) beta-1a is an approved treatment for relapsing remitting multiple sclerosis (RRMS) and has been examined for use in secondary progressive multiple sclerosis (SPMS). However, no information regarding blood transcriptional changes induced by IFN treatment in SPMS patients is available. Our aim was to identify a subgroup of SPMS patients presenting a gene expression signature similar to that of RRMS patients who are clinical responders to IFN treatment. METHODS: SPMS patients (n = 50, 20 IFN treated and 30 untreated) were classified using unsupervised hierarchical clustering according to IFN inducible gene expression profile identified in RRMS clinical responders to treatment. IFN inducible gene expression profile was determined by finding differentially expressed genes (DEGs) between IFN treated (n = 10) and untreated (n = 25) RRMS patients. Validation was performed on an additional independent group of 27 SPMS IFN treated patients by qRT-PCR. RESULTS: One hundred and four DEGs, enriched by IFN signaling pathway (p = 7.4E-08), were identified in IFN treated RRMS patients. Classification of SPMS patients based on these DEGs yielded two patient groups: (1) IFN transcriptional responders (n = 12, 60% of SPMS treated patients) showing gene-expression profile similar to IFN treated RRMS patients; (2) IFN transcriptional non-responders (n = 8) showing expression profile similar to untreated patients. IFN transcriptional responders were characterized by a more active disease, as defined by higher EDSS progression and annual relapse rate. CONCLUSION: Within the IFN treated SPMS population, 60% of patients have a transcriptional response to IFN which is similar to that of RRMS patients who are IFN responders to treatment.