T1 mapping from routine 3D T1-weighted inversion recovery sequences in clinical practice: comparison against reference inversion recovery fast field echo T1 scans and feasibility in multiple sclerosis.

BACKGROUND AND PURPOSE: Quantitative T1 mapping can be an essential tool for assessing tissue injury in multiple sclerosis (MS). We introduce T1-REQUIRE, a method that converts a single high-resolution anatomical 3D T1-weighted Turbo Field Echo (3DT1TFE) scan into a parametric T1 map that could be used for quantitative assessment of tissue damage. We present the accuracy and feasibility of this method in MS. METHODS: 14 subjects with relapsing-remitting MS and 10 healthy subjects were examined. T1 maps were generated from 3DT1TFE images using T1-REQUIRE, which estimates T1 values using MR signal equations and internal tissue reference T1 values. Estimated T1 of lesions, white, and gray matter regions were compared with reference Inversion-Recovery Fast Field Echo T1 values and analyzed via correlation and Bland-Altman (BA) statistics. RESULTS: 159 T1-weighted (T1W) hypointense MS lesions and 288 gray matter regions were examined. T1 values for MS lesions showed a Pearson's correlation of r = 0.81 (p < 0.000), R2 = 0.65, and Bias = 4.18%. BA statistics showed a mean difference of -53.95 ms and limits of agreement (LOA) of -344.20 and 236.30 ms. Non-lesional normal-appearing white matter had a correlation coefficient of r = 0.82 (p < 0.000), R2 = 0.67, Bias = 8.78%, mean difference of 73.87 ms, and LOA of -55.67 and 203.41 ms. CONCLUSIONS: We demonstrate the feasibility of retroactively derived high-resolution T1 maps from routinely acquired anatomical images, which could be used to quantify tissue pathology in MS. The results of this study will set the stage for testing this method in larger clinical studies for examining MS disease activity and progression.

Corrigendum: Adaptive and innate immune responses in multiple sclerosis with anti-CD20 therapy: gene expression and protein profiles.

[This corrects the article DOI: 10.3389/fneur.2023.1158487.].

Inter-rater agreement for the annotation of neurologic signs and symptoms in electronic health records.

The extraction of patient signs and symptoms recorded as free text in electronic health records is critical for precision medicine. Once extracted, signs and symptoms can be made computable by mapping to signs and symptoms in an ontology. Extracting signs and symptoms from free text is tedious and time-consuming. Prior studies have suggested that inter-rater agreement for clinical concept extraction is low. We have examined inter-rater agreement for annotating neurologic concepts in clinical notes from electronic health records. After training on the annotation process, the annotation tool, and the supporting neuro-ontology, three raters annotated 15 clinical notes in three rounds. Inter-rater agreement between the three annotators was high for text span and category label. A machine annotator based on a convolutional neural network had a high level of agreement with the human annotators but one that was lower than human inter-rater agreement. We conclude that high levels of agreement between human annotators are possible with appropriate training and annotation tools. Furthermore, more training examples combined with improvements in neural networks and natural language processing should make machine annotators capable of high throughput automated clinical concept extraction with high levels of agreement with human annotators.

Adaptive and innate immune responses in multiple sclerosis with anti-CD20 therapy: Gene expression and protein profiles.

Background: Anti-CD20 is a highly effective therapy for multiple sclerosis (MS), a disease with multiple abnormalities in function of B and T cells and innate immune cells. Anti-CD20 therapy depletes B cells, which alters antibody production and has diverse effects on B cell immunity. These changes potentially affect immunity beyond B cells in MS. Objective: Determine if anti-CD20 therapy effects non-B cell, as well as B cell, gene expression, and serum protein levels. Methods: Samples were collected from 10 healthy controls and from clinically stable relapsing-remitting MS - 10 untreated, 9 interferon-ß-treated, and 15 ocrelizumab-treated patients were studied before, and 2 weeks and 6 months after, the first anti-CD20 infusion. Peripheral blood mononuclear cells (PBMC) were analyzed with sensitive, 135,000-transcript RNA expression microarrays, using stringent criteria. Gene expression was compared to 43 MS-relevant serum immune and neurotrophic proteins, using multiplex protein assays. Results: Anti-CD20 therapy reduced expression of 413 total genes and 185 B-cell-regulated genes at 2 weeks vs. pre-therapy. Expression of 19 (15%) of these B cell genes returned toward baseline by 6 months, including genes for the B cell activation protein, CD79A, and for immunoglobulin A, D, and G heavy chains. Expression pathways for Th17 and CD4 regulatory T-cell (Treg) development, differentiation, and proliferation also quieted. In contrast, expression increased in Th1 and myeloid cell antiviral, pro-inflammatory, and toll-like receptor (TLR) gene pathways. Conclusion: These findings have clinical implications. B cell gene expression diminishes 2 weeks after anti-CD20 antibody infusion, but begins to rebound by 6 months. This suggests that the optimum time for vaccination is soon before reinfusion of anti-CD20 therapy. In addition, at 6 months, there is enhanced Th1 cell gene expression and induction of innate immune response genes and TLR expression, which can enhance anti-viral and anti-tumor immunity. This may compensate for diminished B cell gene expression after therapy. These data suggest that anti-CD20 therapy has dynamic effect on B cells and causes a compensatory rise in Th1 and myeloid immunity.

Prolonged Interferon-Stimulated Gene and Protein Signatures in Multiple Sclerosis Induced by PEGylated IFN-ß-1a Compared to Non-PEGylated IFN-ß-1a.

Interferon (IFN)-ß-1a (Avonex) and longer half-life, polyethylene glycol-conjugated IFN-ß-1a (PEG-IFN-ß-1a, Plegridy), may generate different molecular responses. We identified different short-term and long-term in vivo global RNA signatures of IFN-stimulated genes in multiple sclerosis (MS) peripheral blood mononuclear cells and in selected paired serum immune proteins. At 6 h, non-PEGylated IFN-ß-1a injection upregulated expression of 136 genes and PEG-IFN-ß-1a upregulated 85. At 24 h, induction was maximal; IFN-ß-1a upregulated 476 genes and PEG-IFN-ß-1a now upregulated 598. Long-term PEG-IFN-ß-1a therapy increased expression of antiviral and immune-regulatory genes (IFIH1, TLR8, IRF5, TNFSF10 [TRAIL], STAT3, JAK2, IL15, and RB1) and IFN signaling pathways (IFNB1, IFNA2, IFNG, IRF7), but downregulated expression of inflammatory genes (TNF, IL1B, and SMAD7). Long-term PEG-IFN-ß-1a induced longer and stronger expression of Th1, Th2, Th17, chemokine, and antiviral proteins than long-term IFN-ß-1a. Long-term therapy also primed the immune system, evoking higher gene and protein induction after IFN reinjection at 7 months than at 1 month of PEG-IFN-ß-1a treatment. Both forms of IFN-ß balanced correlations of expression among these genes and proteins, with positive correlations between Th1 and Th2 families, quelling the "cytokine storm" of untreated MS. Both IFNs induced long-term, potentially beneficial, molecular effects on immune and possibly neuroprotective pathways in MS.

Increased Percentage of CD8+CD28- Regulatory T Cells With Fingolimod Therapy in Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: Fingolimod, an oral therapy for MS, decreases expression of membrane S1P1 receptors on CD4+ memory cells, causing their retention and deactivation in lymph nodes. We determined fingolimod effects on the number and proportion of potentially CNS-damaging CD8+CD28+ cytolytic T lymphocyte cells (CTLs) and on MS-depleted and dysfunctional CD8+CD28- anti-inflammatory suppressor/regulatory T cells (Treg) and on CD8+ T-cell expression of the CD69 activation/lymph node retention protein in MS. METHODS: CD8, CD28, CD4, and CD69 expression on peripheral blood mononuclear cells was measured with flow cytometry. In vitro concanavalin A (ConA) activation of T cells, including CD8+CD28- cells, was used to mimic inflammation. RESULTS: Fifty-nine patients with MS, 35 therapy-naive (16 clinically stable; 19 exacerbating) and 24 fingolimod-treated (19 clinically stable; 5 exacerbating), and 26 matched healthy controls (HCs) were compared. In therapy-naive patients, the CD8+ Treg percent of total lymphocytes was only 1/4 of HC levels. In fingolimod-treated patients, however, CD8+ Treg percentages rose to 2.5-fold higher than in HC and 10-fold higher than in therapy-naive MS. With fingolimod therapy, in contrast, CD8+ CTL levels were less than half of levels in HCs and therapy-naive patients. In HCs and all MS, activation with ConA strongly induced CD69 expression on CD4+ cells and induced 3-fold higher CD69 levels on CD8+ CTL than on CD8+ Treg. Fingolimod and analogs in vitro did not modify lymphocyte CD69 expression. Lower levels of CD69 on CD8+ Treg than on CTL may allow easier Treg egress from lymph nodes and enhance control of peripheral inflammation. In vitro activation reduced the already low CD8+ Treg population in therapy-naive MS, but only slightly altered Treg levels in fingolimod-treated MS. DISCUSSION: Fingolimod therapy markedly increases the percentage of CD8+ Treg in MS, reversing the low CD8+ Treg:CTL ratio seen in untreated MS. The increase in immune regulatory cells has potential therapeutic benefit in MS. Activation in vitro depletes CD8+CD28+CTL in patients with MS; the loss is more pronounced in older patients with MS. This suggests that inflammation can disrupt the tenuous immune regulation in MS, especially in older patients.

Subtypes of relapsing-remitting multiple sclerosis identified by network analysis.

We used network analysis to identify subtypes of relapsing-remitting multiple sclerosis subjects based on their cumulative signs and symptoms. The electronic medical records of 113 subjects with relapsing-remitting multiple sclerosis were reviewed, signs and symptoms were mapped to classes in a neuro-ontology, and classes were collapsed into sixteen superclasses by subsumption. After normalization and vectorization of the data, bipartite (subject-feature) and unipartite (subject-subject) network graphs were created using NetworkX and visualized in Gephi. Degree and weighted degree were calculated for each node. Graphs were partitioned into communities using the modularity score. Feature maps visualized differences in features by community. Network analysis of the unipartite graph yielded a higher modularity score (0.49) than the bipartite graph (0.25). The bipartite network was partitioned into five communities which were named fatigue, behavioral, hypertonia/weakness, abnormal gait/sphincter, and sensory, based on feature characteristics. The unipartite network was partitioned into five communities which were named fatigue, pain, cognitive, sensory, and gait/weakness/hypertonia based on features. Although we did not identify pure subtypes (e.g., pure motor, pure sensory, etc.) in this cohort of multiple sclerosis subjects, we demonstrated that network analysis could partition these subjects into different subtype communities. Larger datasets and additional partitioning algorithms are needed to confirm these findings and elucidate their significance. This study contributes to the literature investigating subtypes of multiple sclerosis by combining feature reduction by subsumption with network analysis.

Anti-CD20 therapy corrects a CD8 regulatory T cell deficit in multiple sclerosis.

OBJECTIVE: To determine the effect of long-term anti-CD20 B-cell-depleting treatment on regulatory T cell immune subsets that are subnormal in untreated MS patients. METHODS: 30 clinically stable MS patients, before and over 38 months of ocrelizumab treatment, were compared to 13 healthy controls, 29 therapy-naïve MS, 9 interferon-ß-treated MS, 3 rituximab-treated MS, and 3 rituximab-treated patients with other autoimmune inflammatory diseases. CD8, CD28, CD4, and FOXP3 expression in peripheral blood mononuclear cells was quantitated with flow cytometry. RESULTS: CD8+ CD28- regulatory cells rose from one-third of healthy control levels before ocrelizumab treatment (2.68% vs 7.98%), normalized by 12 months (13.5%), and rose to 2.4-fold above healthy controls after 18 months of ocrelizumab therapy (19.0%). CD4+ FOXP3+ regulatory cells were lower in MS than in healthy controls (7.98%) and showed slight long-term decreases with ocrelizumab. CD8+ CD28- and CD4+ FOXP3+ regulatory T cell percentages in IFN-ß-treated MS patients were between those of untreated MS and healthy controls. INTERPRETATION: Long-term treatment with ocrelizumab markedly enriches CD8+ CD28- regulatory T cells and corrects the low levels seen in MS before treatment, while slightly decreasing CD4+ FOXP3+ regulatory T cells. Homeostatic enrichment of regulatory CD8 T cells provides a mechanism, in addition to B cell depletion, for the benefits of anti-CD20 treatment in MS.

Modulation of immune responses by bile acid receptor agonists in myasthenia gravis.

Bile acids bind to multiple receptors, including Takeda G protein-coupled receptor 5 (TGR5) and farnesoid-X-receptors alpha (FXRα). We compared the response of PBMCs to the activation of these receptors in healthy controls and myasthenic patients. We found that TGR5 is a more potent negative regulator of T cell cytokine response than FXRα in both groups. In contrast, TGR5 and FXRα agonists elicit distinct B cell responses in myasthenia compared to controls, specifically on the frequency of IL-6+ B cells and regulatory B cells, as well as IL-10 secretion from PBMCs. We propose that TGR5 is a potential therapeutic target in myasthenia.

Vitamin D enhances responses to interferon-ß in MS.

OBJECTIVE: To determine the effect of vitamin D3 on interferon-ß (IFN-ß) response and immune regulation in MS mononuclear cells (MNCs). METHODS: MNCs from 126 subjects, including therapy-naive patients with different forms of MS, plus patients with MS receiving IFN-ß or glatiramer treatment, plus healthy controls were incubated in vitro with IFN-ß-1b ± vitamin D3 (calcitriol). Activation of the IFN-ß-induced transcription factor, p-Y-STAT1, and antiviral myxovirus A (MxA) protein was measured with flow cytometry and Western blots; serum proteins were measured with a customized 31-protein multiplex assay. RESULTS: Vitamin D enhanced in vitro IFN responses, as measured by induction of p-Y-STAT1 and MxA in MNCs, T cells, and monocytes. Vitamin D augmentation of IFN responses was seen in untreated and in IFN-ß-1b-treated MS. The combination of vitamin D plus IFN-ß reduced Th1 and Th17 cytokines, and increased Th2 responses, reversing the effect of IFN-ß alone. Exacerbations and progression in untreated patients reduced the vitamin D enhancement of IFN responses. Vitamin D had less effect on IFN response in clinically stable glatiramer-treated than in IFN-ß-treated patients. CONCLUSION: Vitamin D enhances IFN-ß induction of multiple proteins and also reverses the Th1/Th2 bias in MS seen with IFN-ß alone. The combination of vitamin D and IFN-ß has potential benefit in ameliorating MS.