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Autoimmune encephalitis (AIE) encompasses a range of inflammatory disorders manifesting with some combination of encephalopathy, seizures, behavioral changes, movement disorders, dysautonomia or other neurologic symptoms. Anti- N -methyl- d -aspartate receptor encephalitis (NMDARE) is the most common AIE and is an autoantibody mediated disorder, often paraneoplastic. Untreated or undertreated AIE has a high degree of morbidity and mortality. Immunosuppressive treatment regimens including glucocorticoids, plasma exchange (PLEX), intravenous immunoglobulin (IVIG) and rituximab used alone or in combination for such patients. Patients' refractory to such treatments requires more aggressive and potentially toxic therapies. We report favorable outcomes in patients with refractory AIE who received intrathecal methotrexate (IT-MTX) as part of treatment. Cases at our institution seen between 2010 and 2020 were reviewed. We identified 5 patients in our clinical practice whose clinical presentation was compatible with NMDARE. Three patients met criteria for definite NMDARE. An additional two patients met criteria for probable NMDARE in the acute setting but were ultimately seronegative autoimmune encephalitis. All patients received at least one dose of IT-MTX after failing conventional therapies. At the time of IT-MTX administration patients were catatonic, comatose, or severely encephalopathic despite initial treatments. All patients were treated with methylprednisolone; 3 received a course of IVIG, 4 underwent PLEX, and 4 received rituximab. At the time IT-MTX was given, three patients required mechanical ventilation and 1 had a pacemaker placed for autonomic failure. Two patients were under consideration for transition to palliative care. All patients improved and were at or near their premorbid baseline at last follow-up. All patients tolerated IT-MTX well. This retrospective review demonstrates the efficacy of intrathecal methotrexate in the treatment of severe AIE who had failed other immunosuppressive regimens. • Treatment options for refractory autoimmune encephalitis are limited. • Intrathecal methotrexate appears to be effective and well tolerated. • Intrathecal methotrexate is a potentially underutilized treatment option in severe and refractory autoimmune encephalitis. [ABSTRACT FROM AUTHOR]

Given the known neuroreparative actions of IL-33 in experimental models of central nervous system (CNS) injury, we predicted that compounds which induce IL-33 are likely to promote remyelination. We found anacardic acid as a candidate molecule to serve as a therapeutic agent to promote remyelination. Addition of anacardic acid to cultured oligodendrocyte precursor cells (OPCs) rapidly increased expression of myelin genes and myelin proteins, suggesting a direct induction of genes involved in myelination by anacardic acid. Also, when added to OPCs, anacardic acid resulted in the induction of IL-33. In vivo, treatment of with anacardic acid in doses which ranged from 0.025 mg/kg to 2.5 mg/kg, improved pathologic scores in experimental allergic encephalitis (EAE) and in the cuprizone model of demyelination/remyelination. Electron microscopic studies performed in mice fed with cuprizone and treated with anacardic acid showed lower g-ratio scores when compared to controls, suggesting increased remyelination of axons. In EAE, improvement in paralytic scores was seen when the drug was given prior to or following the onset of paralytic signs. In EAE and in the cuprizone model, areas of myelin loss, which are likely to remyelinate, was associated with a greater recruitment of IL-33-expressing OPCs in mice which received anacardic acid when compared to controls. [ABSTRACT FROM AUTHOR]

Various sensors that detect double-stranded RNA, presumably of viral origin, exist in eukaryotic cells and induce IFN-responses. Ongoing IFN-responses have also been documented in a variety of human autoimmune diseases including relapsing-remitting multiple sclerosis (RRMS) but their origins remain obscure. We find increased IFN-responses in leukocytes in relapsing-remitting multiple sclerosis at distinct stages of disease. Moreover, endogenous RNAs isolated from blood cells of these same patients recapitulate this IFN-response if transfected into naïve cells. These endogenous RNAs are double-stranded RNAs, contain Alu and Line elements and are transcribed from leukocyte transcriptional enhancers. Thus, transcribed endogenous retrotransposon elements can co-opt pattern recognition sensors to induce IFN-responses in RRMS. • Type 1 interferon responses are elevated at distinct stages of relapsing remitting multiple sclerosis. • Endogenous RNAs isolated from blood cells of these same patients recapitulate this type 1 IFN response if transfected into naïve cells. • These endogenous RNAs are double-‐stranded RNAs, contain Alu and Line elements and are transcribed from leukocyte transcriptional enhancers. • In vitro transcribed Alu RNAs stimulate type 1 interferon responses. • Endogenous Alu and Line double stranded RNAs may be the source of type 1 IFN responses in relapsing remitting multiple sclerosis and possibly other human autoimmune diseases. One sentence summary: Endogenous double-stranded RNAs containing Alu and Line elements transcribed from transcriptional enhancers stimulate IFN-responses in relapsing remitting multiple sclerosis. [ABSTRACT FROM AUTHOR]

Small ubiquitin like modifiers (SUMO) are reversible posttranslational modifiers of intracellular proteins. In the CNS, expression of myelin genes is regulated by state of SUMOylation of their respective transcription factors. In the immune system, deSUMOylation activates innate immune responses and promotes anti-viral immunity. However, the role played by SUMO in an adaptive immune response and in the development of T cell mediated autoimmune disease has not been previously described. TAK981 is a synthetic small molecule which by forming adducts with SUMO proteins prevents SUMOylation. We examined the expression of myelin genes and their transcription factors following culture with TAK981 in Oligodendrocyte Precursor Cells (OPC). We found that myelin basic protein (MBP), a key myelin protein, is upregulated in OPC in the presence of TAK981. We also found increased expression of transcription factors Sox 1 0 and Myrf, which engage in the expression of MBP. In the Cuprizone model of demyelination/remyelination, animals which were treated with TAK981 showed increased remyelination in areas of demyelination and an increase in the number of maturing oligodendrocytes compared to vehicle treated controls. In in vitro cultures of lymphocytes, TAK981 reduced the expression of TH17 in T cells in mice immunized with MOGp35–55. Following in vivo treatment with TAK981, there was a significant reduction in the clinical and pathological severity in mice immunized to develop experimental allergic encephalitis (EAE). The dual effects of deSUMOylation on remyelination and in regulating an autoimmune adaptive response offers a novel approach to the management of human inflammatory demyelinating diseases such as multiple sclerosis. • Small ubiquitin like modifiers (SUMO) are reversible posttranslational modifiers of intracellular proteins. • Inhibition of SUMOylation increases the expression of myelin genes and inhibits TH17 expression • TAK981 is a synthetic small molecule which by forming adducts with SUMO proteins prevents SUMOylation. • TAK981 when administered in vivo improves myelination in Cuprizone model of CNS demyelination/remyelination • TAK981 also reduces the expression of IL-17 and ameliorates TH17 mediated autoimmune encephalomyelitis [ABSTRACT FROM AUTHOR]

Background and Purpose: An imaging biomarker of myelin integrity is an unmet need in multiple sclerosis (MS). Selective inversion recovery (SIR) quantitative magnetization transfer imaging (qMT) provides assays of myelin content in the human brain. We previously translated the SIR method to 7T and incorporated a rapid turbo field echo (TFE) readout for whole-brain imaging within clinically acceptable scan times. We herein provide histological validation and test in vivo feasibility and applicability of the SIR-TFE protocol in MS.Methods: Clinical (T1 - and T2 -weighted) and SIR-TFE MRI scans were performed at 7T in a postmortem MS brain and MRI data were acquired in 10 MS patients and 14 heathy volunteers in vivo. The following parameters were estimated from SIR data: the macromolecular-to-free water pool-size-ratio (PSR), the spin-lattice relaxation rate of water (R1f ), and the MT exchange rate (kmf ). Differences in SIR parameters across tissue types, eg, white matter lesions (WM-Ls) and normal appearing WM (NAWM) in patients, and normal white matter (NWM) in heathy volunteers were evaluated. Associations between SIR parameters and disability scores were assessed.Results: For postmortem scans, correspondence was observed between WM-Ls and NAWM from histology and PSR/R1f values. In vivo differences were detected for PSR, R1f , and kmf between WM-Ls and NWM (P ≤ .041). Associations were seen between WM-Ls/ NAWM PSR and disability scores (r ≤ -.671, P ≤ .048).Conclusions: SIR-qMT at 7T provides sensitive, quantitative measures of myelin integrity for clinical and research applications. [ABSTRACT FROM AUTHOR]

Patients with multiple sclerosis present with focal lesions throughout the spinal cord. There is a clinical need for non-invasive measurements of spinal cord activity and functional organization in multiple sclerosis, given the cord's critical role in the disease. Recent reports of spontaneous blood oxygenation level-dependent fluctuations in the spinal cord using functional MRI suggest that, like the brain, cord activity at rest is organized into distinct, synchronized functional networks among grey matter regions, likely related to motor and sensory systems. Previous studies looking at stimulus-evoked activity in the spinal cord of patients with multiple sclerosis have demonstrated increased levels of activation as well as a more bilateral distribution of activity compared to controls. Functional connectivity studies of brain networks in multiple sclerosis have revealed widespread alterations, which may take on a dynamic trajectory over the course of the disease, with compensatory increases in connectivity followed by decreases associated with structural damage. We build upon this literature by examining functional connectivity in the spinal cord of patients with multiple sclerosis. Using ultra-high field 7 T imaging along with processing strategies for robust spinal cord functional MRI and lesion identification, the present study assessed functional connectivity within cervical cord grey matter of patients with relapsing-remitting multiple sclerosis (n = 22) compared to a large sample of healthy controls (n = 56). Patient anatomical images were rated for lesions by three independent raters, with consensus ratings revealing 19 of 22 patients presented with lesions somewhere in the imaged volume. Linear mixed models were used to assess effects of lesion location on functional connectivity. Analysis in control subjects demonstrated a robust pattern of connectivity among ventral grey matter regions as well as a distinct network among dorsal regions. A gender effect was also observed in controls whereby females demonstrated higher ventral network connectivity. Wilcoxon rank-sum tests detected no differences in average connectivity or power of low frequency fluctuations in patients compared to controls. The presence of lesions was, however, associated with local alterations in connectivity with differential effects depending on columnar location. The patient results suggest that spinal cord functional networks are generally intact in relapsing-remitting multiple sclerosis but that lesions are associated with focal abnormalities in intrinsic connectivity. These findings are discussed in light of the current literature on spinal cord functional MRI and the potential neurological underpinnings. [ABSTRACT FROM AUTHOR]

Abstract: Background: It is not known whether the currently available treatment regimen of interferon beta-1b (IFN²-1b) 250 µg provides the maximum benefit possible in the treatment of relapsing-remitting multiple sclerosis (RRMS), or whether higher doses of IFN²-1b will prove to be more beneficial. Objective: The objective of the present study was to evaluate the tolerability and safety profile of IFN²-1b 500 µg compared with the currently approved 250-µg dose. Methods: A multicenter, randomized, double-blind, parallel-group pilot study was carried out to compare IFN²-1b 250 µg with IFN²-1b 500 µg, both self-administered SC QOD for ≥ 12 weeks in patients with RRMS. Patients in both groups started with 25% (0.25 mL) of their final dose and were scheduled to increase the dose by 0.25 mL every 2 weeks, so that the full dose (1.0 mL, 250 µg or 500 µg) would be reached by week 7. The primary outcome measure was the percentage of patients experiencing each of the following adverse events (AEs): influenza-like symptoms (general term used to code the presence of>1 symptom typical of influenza), fever, myalgia, asthenia, headache, injection-site reactions, injection-site pain, or liver or hematologic abnormalities. All patients underwent a priori magnetic resonance imaging (MRI) with 0.1 mmol/kg gadolinium (Gd)-diethylenetriaminepentaacetic acid as contrast medium at screening and at week 12. MRI evaluation was included as a safety measure to monitor for excessive new disease not visible through clinical symptoms. Results: Seventy-seven patients were assessed for inclusion in the study. Of these, 6 patients were screening failures and the remaining 71 were randomized to treatment (38–250 and 33–500 µg IFN²-1b). The uneven numbers in the groups were a consequence of the randomization process. Two patients in the 250-µg group (withdrawal of consent) and 1 in the 500-µg group (not completing follow-up visit) prematurely discontinued medication. The demographic characteristics were not significantly different between the 250-µg (n = 38; mean [SD] age, 37.9 [8.3] years; weight, 83.5 [19.0] kg; height, 168.4 [9.3] cm) and 500-µg (n = 33; mean [SD] age, 37.8 [7.7] years; weight, 82.3 [19.5] kg; height, 169.9 [10.5] cm) treatment groups. The patients in both groups were mostly white (87% and 73%, respectively). Baseline Expanded Disability Status Scale scores also were not significantly different between the 2 groups (mean [SD] score, 2.8 [1.4] vs 2.0 [1.4], respectively). In the IFN²-1b 250-µg group, 97% of the patients titrated to the full dose at some point during the course of the study, compared with 91% of the 500-µg group (P = NS). A dose-response effect was observed in some of the more frequent AEs (no. [%]) that included influenza-like syndrome (250-µg group, 13 [34] vs 500-µg group, 16 [48]), asthenia (13 [34] vs 16 [48], respectively), headache (12 [32] vs 12 [36]), myalgia (10 [26] vs 13 [39]), hypesthesia (10 [26] vs 11 [33]), nausea (6 [16] vs 8 [24]), paresthesia (6 [16] vs 8 [24]), myasthenia (4 [11] vs 8 [24]), chills (3 [8] vs 6 [18]), depression (3 [8] vs 5 [15]), back pain (2 [5] vs 5 [15]), increased liver enzymes (4 [11] vs 6 [18]), lymphopenia (4 [11] vs 3 [9]), fever (2 [5] vs 4 [12]), and pain in extremities (1 [3] vs 4 [12]). The between-group incidence of injection-site reactions was not significantly different. No new or unexpected AEs were recorded. Changes in MRI parameters between screening and 12 weeks were not significantly different between dose groups; these included median T2 lesion volume, median Gd-enhancedlesionvolume,medianGd-enhancedlesion number, and mean number of newly active lesions. Conclusions: IFN²-1b 500 µg administered SC QOD was generally well tolerated in these patients with RRMS. Large, randomized controlled studies are needed to determine if there are significant differences in MRI end points between the 250- and 500-µg doses. [Copyright &y& Elsevier]

Objective: We tested the hypothesis that the expression of IL-33 in MS is dynamic and is likely to reflect the clinical and radiological changes during the course of RRMS., Methods: MS with either clinical or radiological relapses were recruited for the study and followed for one year. IL-33 and a panel of genes was measured by q PCR and flow cytometry at different time points., Results: Among 22 RRMS patients, 4 patients showed highest levels of IL-33 at the time they were recruited to the study (Month 0); in 14 patients highest levels of IL-33 were seen between 6-11 months after relapse and in 4 patients maximal levels of IL-33 were seen 12 months after relapse. A similar pattern of IL-33 kinetics was seen when IL-33 was measured by flow cytometry in an additional cohort of 12 patients. The timing of the improvement clinically did not correlate with IL-33 expression with highest expression levels either preceding or following clinical recovery. From our whole genome RNA-sequencing data we found a strong correlation between expression levels of IL-33 and a ~2000 mRNA genes. However, none of these genes encoded proteins involved in either innate or adaptive immunity. Rather, many of the genes that correlated highly with IL-33 encoded to proteins involved in DNA repair or mitochondrial function and mRNA splicing pathways., Interpretation: Given the neuro-reparative and remodeling functions attributed to IL-33, it is likely that some of the novel genes we have uncovered may be involved in repair and recovery of the CNS in MS., Competing Interests: We have the following interests. The study was funded in part by Serono and IQuity Inc. IQuity is the employer of Guzel Shaginurova and John T. Tossberg. JTT, CFS and TMA are shareholders in IQuity, Inc., a data analytics company that seeks to create novel diagnostic tests for inflammatory diseases including multiple sclerosis. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

The relationship between the p53 signal pathway and the response of human peripheral blood mononuclear cells (PBMC) to interferon (IFN)-beta has hitherto not been examined. Using an oligonucleotide microarray, we found differential expression of at least 70 genes involved in the p53 signal pathway, including p53, which regulate cell proliferation and cell death following stimulation with IFN-beta. We verified our observations on a limited set of p53-regulated genes at the transcriptional and translational levels. We also examined the consequences of the activation of the p53 signal pathway by IFN-beta in PBMC. When cultured in the presence of T-cell mitogens, IFN-beta restricted the entry of lymphocytes from the G0/G1 phase to the S phase and reduced the number of cells in the G2 phase. The addition of IFN-beta alone did not increase apoptosis. However, in the presence of actinomycin D, a DNA-damaging agent, addition of IFN-beta enhanced the susceptibility of PBMC to apoptosis. These observations suggest that, in spite of the activation of a number of mutually overlapping pathways mediating cell death, cell cycle arrest was the most evident consequence of IFN-beta signalling in PBMC.

Hypoxia-inducible factor-1alpha (HIF-1alpha) is a transcription factor induced under hypoxic conditions. HIF-1alpha promotes the expression of genes encoding proteins that increase the cellular supply of oxygen and promote survival in periods of cellular stress and availability of cellular energy. We examined the effect of desferrioxamine (DFO) and cobalt chloride (CoCl(2)), two agents known to increase the stability of HIF-1alpha, and its effect on the survivability of an oligodendroglial cell line, MO3.13, when cultured with tumor necrosis factor-alpha (TNFalpha). Our studies showed that, unlike a murine microglial cell line (BV-2), MO3.13 cells do not induce HIF-1alpha in the presence of TNFalpha. MO3.13 cells do stabilize HIF-1alpha in the presence of DFO or CoCl(2). When MO3.13 cell were preconditioned with either DFO or CoCl(2), addition of TNFalpha further increased protein levels of HIF-1alpha. The mechanisms that underlie the increase in protein levels of HIF -1alpha seen, following addition of TNFalpha in preconditioned cells is due to an increase in transcription of the HIF-1alpha gene. Increased cellular levels of HIF-1alpha is associated with improved survival of MO3.13 cells, when cultured with TNFalpha after a period of preconditioning by DFO or CoCl(2). These studies suggest that compoundsthat increase HIF-1alpha can function as neuroprotective agents in inflammatory disorders of the CNS.

Microglia, the resident macrophage of the brain, mediates immune and inflammatory responses in the central nervous system (CNS). Activation of microglia and secretion of inflammatory cytokines associate with the pathogenesis of CNS diseases, including multiple sclerosis (MS), Alzheimer's disease (AD), Parkinson's disease, prion disease, and AIDS dementia. Microbial pathogens, cytokines, chemokines, and costimulatory molecules are potent inducers of microglial activation in the CNS. Signaling through its receptor, IL-3 induces the activation of JAK-STAT and MAP kinase pathways in microglial cells. In this study, we found that in vitro treatment of EOC-20 microglial cells with tyrphostin AG490 blocked IL-3-induced tyrosine phosphorylation of JAK2, STAT5A, and STAT5B signaling proteins. Stable transfection of EOC-20 cells with a dominant negative JAK2 mutant also blocked IL-3-induced tyrosine phosphorylation of JAK2, STAT5A, and STAT5B in microglia. The blockade of JAK2-STAT5 pathway resulted in a decrease in IL-3-induced proliferation and expression of CD40 and major histocompatibility complex class II molecules in microglia. These findings highlight the fact that JAK2-STAT5 signaling pathway plays a critical role in mediating IL-3-induced activation of microglia., (Copyright 2003 Wiley-Liss, Inc.)

There is considerable controversy concerning the evidence for the presence of Chlamydia pneumoniae in the cerebrospinal fluid (CSF) of both multiple sclerosis (MS) patients and patients with other neurological diseases (OND). In order to clarify this issue, the laboratories at Vanderbilt University Medical Center (VUMC) and the University of South Florida (USF) examined the reproducibility of their respective PCR assays for the detection of C. pneumoniae DNA in the CSF of a common group of MS patients and OND controls. The two laboratories used different DNA extraction and PCR techniques in order to determine the prevalence of the C. pneumoniae genome in both monosymptomatic and clinically definite MS patients as well as in OND controls. In clinically definite MS patients, the VUMC and USF detection rates were 72 and 61%, respectively, and in patients with monosymptomatic MS, the VUMC and USF detection rates were 41 and 54%, respectively. The PCR signal was positive for 7% of the OND controls at VUMC and for 16% at USF. These studies confirm our previous reports concerning the high prevalence of C. pneumoniae in the CSF of MS patients. The presence of C. pneumoniae in patients with monosymptomatic MS would also suggest that infection with the organism occurs early in the course of the disease.