ObjectiveTo determine the effect of vitamin D3 on interferon-β (IFN-β) response and immune regulation in MS mononuclear cells (MNCs).MethodsMNCs from 126 subjects, including therapy-naive patients with different forms of MS, plus patients with MS receiving IFN-β or glatiramer treatment, plus healthy controls were incubated in vitro with IFN-β-1b ± vitamin D3 (calcitriol). Activation of the IFN-β–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.ResultsVitamin 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-β-1b–treated MS. The combination of vitamin D plus IFN-β reduced Th1 and Th17 cytokines, and increased Th2 responses, reversing the effect of IFN-β 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-β–treated patients.ConclusionVitamin D enhances IFN-β induction of multiple proteins and also reverses the Th1/Th2 bias in MS seen with IFN-β alone. The combination of vitamin D and IFN-β has potential benefit in ameliorating MS.

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-β-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-β-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.

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 clinical concepts in an ontology. Extracting clinical concepts 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.

Background and ObjectivesFingolimod, 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.MethodsCD8, 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.ResultsFifty-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.DiscussionFingolimod 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.