Neoehrlichia mikurensis-An emerging opportunistic tick-borne infection in immunosuppressed patients.

BACKGROUND: Neoehrlichia mikurensis (N. mikurensis) is a newly discovered tick-borne pathogen that can inflict life-threatening illness in immunocompromised patients. N. mikurensis infection is only detectable by polymerase chain reaction (PCR)-based methodologies. We describe three distinct clinical manifestations of N. mikurensis infection (neoehrlichiosis) in Danish patients receiving B-lymphocyte-depleting therapy, rituximab, for underlying hematological, rheumatological, or neurological disorders. All three patients went through a protracted pre-diagnostic period. METHODS: N. mikurensis DNA was detected and confirmed using two methods. Blood was tested by specific real-time PCR targeting the groEL gene and by 16S and 18S profiling followed by sequencing. Bone marrow was analyzed by 16S and 18S profiling. RESULTS: N. mikurensis was detected in blood samples in all three cases and in bone marrow from one of the three. The severity of the symptoms ranged from prolonged fever lasting more than 6 months to life-threatening hyperinflammation in the form of hemophagocytic lymphohistiocytosis (HLH). Interestingly, all patients presented with splenomegaly and two with hepatomegaly. After starting doxycycline therapy, symptoms were relieved within a few days, and biochemistry and organomegaly quickly normalized. CONCLUSION: We present three Danish patients recognized by the same clinician over a period of 6 months, strongly suggesting that many cases are going unrecognized. Second, we describe the first case of N. mikurensis-induced HLH and emphasize the potential severity of undetected neoehrlichiosis.

Relationship between soluble CD25 and gene expression in healthy individuals and patients with multiple sclerosis.

Genome wide association studies and fine mapping has established a firm link between the IL2RA gene, encoding the interleukin-2 receptor α-chain CD25, and susceptibility to multiple sclerosis (MS). We hypothesized that gene expression in peripheral blood mononuclear cells (PBMCs) from healthy controls (HCs) and MS patients are associated with IL2RA SNP rs2104286 and that gene expression levels correlate with soluble CD25 (sCD25) concentrations - that are affected by rs2104286. We used the Affymetrix Human Gene ST 1.0 microarray to analyze gene expression levels in PBMCs from 18 HCs and 51MS patients. Plasma concentrations of sCD25 were measured by ELISA in all individuals. In HCs 266 genes correlated with sCD25 with Spearman's rho≥0.707; 70 of these genes had a false discovery rate (FDR) value of q<0.05. These genes were highest expressed in cells belonging to the innate immune system. Gene-networks were focused around NFKB1, TNF, BCL6 and STAT1. Eighteen genes correlated with sCD25 with rho≥0.707 in relapsing remitting MS versus 33 in secondary progressive and 34 in primary progressive MS. None had a FDR<0.05. Thirty-eight and 23 genes were differentially expressed between rs2104286 genotype-groups in MS patients and HCs respectively, however they were not significant after FDR correction. Our study indicates that rs2104286 influences gene expression in PBMCs in HCs as shown by the high correlations with the rs2104286-affected sCD25 protein. Correlations were strongest in HCs suggesting that immunological alterations may obscure the role of the IL2RA SNP rs2104286 in established MS.

Study protocol: effects of exercise booster sessions on preservation of exercise-induced adaptations in persons with multiple sclerosis, a multicentre randomised controlled trial-the MS BOOSTER trial.

INTRODUCTION: Multiple sclerosis (MS) causes a broad range of symptoms, with physical function being one of the most disabling consequences according to patients themselves. Exercise effectively improves lower extremity physical function. Nonetheless, it is unknown which exercise modality is most effective and it remains challenging to keep persons with MS adhering to exercise over a longer period. Therefore, the present study aims to investigate how exercise booster sessions (EBS) influence the sustainability of exercise-induced effects on physical function, and furthermore, to investigate which exercise modality (aerobic training or resistance training) is most effective in terms of improving physical function. MATERIALS AND METHODS: This study is a multi-arm, parallel-group, open-label multicentre randomised controlled trial investigating the effects of EBS. Participants (n=150) are initially randomised to 12 weeks of either resistance training+usual care, aerobic training+usual care or usual care. After 12 weeks of intervention, participants in the exercise groups will again be randomised to either EBS+usual care or usual care during a 40-week follow-up period. The primary outcome is physical function (composite score based on 6-min walk test and five-time sit to stand), and the secondary outcomes are fatigue, cognition, physical activity, symptoms of depression and quality of life. ETHICS AND DISSEMINATION: The study is approved by the Central Denmark Region Committees on Health Research Ethics (1-10-72-237-21) and is registered at the Danish Data Protection Agency (2016-051-000001) and at Clinicaltrials.gov (NCT04913012). All study findings will be published in scientific peer-reviewed journals and presented at scientific conferences. TRIAL REGISTRATION NUMBER: NCT04913012.

Natalizumab treatment of multiple sclerosis - a Danish nationwide study with 13 years of follow-up.

BACKGROUND: Natalizumab is a widely used high-efficacy treatment in multiple sclerosis (MS). Real-world evidence regarding long-term effectiveness and safety is warranted. We performed a nationwide study evaluating prescription patterns, effectiveness, and adverse events. METHODS: A nationwide cohort study using the Danish MS Registry. Patients initiating natalizumab between June 2006 and April 2020 were included. Patient characteristics, annualized relapse rates (ARRs), confirmed Expanded Disability Status Scale (EDSS) score worsening, MRI activity (new/enlarging T2- or gadolinium-enhancing lesions), and reported adverse events were evaluated. Further, prescription patterns and outcomes across different time periods ("epochs") were analysed. RESULTS: In total, 2424 patients were enrolled, with a median follow-up time of 2.7 years (interquartile range (IQR) 1.2-5.1). In recent epochs, patients were younger, had lower EDSS scores, had fewer pre-treatment relapses and were more often treatment naïve. At 13 years of follow-up, 36% had a confirmed EDSS worsening. On-treatment ARR was 0.30, corresponding to a 72% reduction from pre-initiation. MRI activity was rare, 6.8% had activity within 2-14 months from treatment start, 3.4% within 14-26 months, and 2.7% within 26-38 months. Approximately 14% of patients reported adverse events, with cephalalgia constituting the majority. During the study, 62.3% discontinued treatment. Of these, the main cause (41%) was due to JCV antibodies, while discontinuations due to disease activity (9%) or adverse events (9%) were less frequent. CONCLUSION: Natalizumab is increasingly used earlier in the disease course. Most patients treated with natalizumab are clinically stable with few adverse events. JCV antibodies constitute the main cause for discontinuation.

Imaging cortical multiple sclerosis lesions with ultra-high field MRI.

BACKGROUND: Cortical lesions are abundant in multiple sclerosis (MS), yet difficult to visualize in vivo. Ultra-high field (UHF) MRI at 7 T and above provides technological advances suited to optimize the detection of cortical lesions in MS. PURPOSE: To provide a narrative and quantitative systematic review of the literature on UHF MRI of cortical lesions in MS. METHODS: A systematic search of all literature on UHF MRI of cortical lesions in MS published before September 2020. Quantitative outcome measures included cortical lesion numbers reported using 3 T and 7 T MRI and between 7 T MRI sequences, along with sensitivity of UHF MRI towards cortical lesions verified by histopathology. RESULTS: 7 T MRI detected on average 52 ± 26% (mean ± 95% confidence interval) more cortical lesions than the best performing image contrast at 3 T, with the largest increase in type II-IV intracortical lesion detection. Across all studies, the mean cortical lesion number was 17 ± 6 per patient. In progressive MS cohorts, approximately four times more cortical lesions were reported than in CIS/early RRMS, and RRMS. Yet, there was no difference in lesion type ratio between these MS subtypes. Furthermore, superiority of one MRI sequence over another could not be established from available data. Post-mortem lesion detection with UHF MRI agreed only modestly with pathological examinations. Mean pro- and retrospective sensitivity was 33 ± 6% and 71 ± 10%, respectively, with the highest sensitivity towards type I and type IV lesions. CONCLUSION: UHF MRI improves cortical lesion detection in MS considerably compared to 3 T MRI, particularly for type II-IV lesions. Despite modest sensitivity, 7 T MRI is still capable of visualizing all aspects of cortical lesion pathology and could potentially aid clinicians in diagnosing and monitoring MS, and progressive MS in particular. However, standardization of acquisition and segmentation protocols is needed.

Transcriptome and Function of Novel Immunosuppressive Autoreactive Invariant Natural Killer T Cells That Are Absent in Progressive Multiple Sclerosis.

BACKGROUND AND OBJECTIVE: The aim of this study was to determine whether natural killer T (NKT) cells, including invariant (i) NKT cells, have clinical value in preventing the progression of multiple sclerosis (MS) by examining the mechanisms by which a distinct self-peptide induces a novel, protective invariant natural killer T cell (iNKT cell) subset. METHODS: We performed a transcriptomic and functional analysis of iNKT cells that were reactive to a human collagen type II self-peptide, hCII707-721, measuring differentially induced genes, cytokines, and suppressive capacity. RESULTS: We report the first transcriptomic profile of human conventional vs novel hCII707-721-reactive iNKT cells. We determined that hCII707-721 induces protective iNKT cells that are found in the blood of healthy individuals but not progressive patients with MS (PMS). By transcriptomic analysis, we observed that hCII707-721 promotes their development and proliferation, favoring the splicing of full-length AKT serine/threonine kinase 1 (AKT1) and effector function of this unique lineage by upregulating tumor necrosis factor (TNF)-related genes. Furthermore, hCII707-721-reactive iNKT cells did not upregulate interferon (IFN)-γ, interleukin (IL)-4, IL-10, IL-13, or IL-17 by RNA-seq or at the protein level, unlike the response to the glycolipid alpha-galactosylceramide. hCII707-721-reactive iNKT cells increased TNFα only at the protein level and suppressed autologous-activated T cells through FAS-FAS ligand (FAS-FASL) and TNFα-TNF receptor I signaling but not TNF receptor II. DISCUSSION: Based on their immunomodulatory properties, NKT cells have a potential value in the treatment of autoimmune diseases, such as MS. These significant findings suggest that endogenous peptide ligands can be used to expand iNKT cells, without causing a cytokine storm, constituting a potential immunotherapy for autoimmune conditions, including PMS.

Dimethyl fumarate therapy reduces memory T cells and the CNS migration potential in patients with multiple sclerosis.

BACKGROUND: Dimethyl fumarate (DMF) is a disease-modifying therapy for patients with relapsing-remitting multiple sclerosis (RRMS). T cells are major contributors to the pathogenesis of RRMS, where they regulate the pathogenic immune response and participate in CNS lesion development. OBJECTIVES: In this study we evaluate the therapeutic effects of DMF on T cell subpopulations, their CNS migration potential and effector functions. METHODS: Blood and CSF from untreated and DMF-treated patients with RRMS and healthy donors were analyzed by flow cytometry. RESULTS: DMF reduced the prevalence of circulating proinflammatory CD4+ and CD8+ memory T cells, whereas regulatory T cells were unaffected. Furthermore, DMF reduced the frequency of CD4+ T cells expressing CNS-homing markers. In coherence, we found a reduced recruitment of CD4+ but not CD8+ T cells to CSF. We also found that monomethyl fumarate dampened T cell proliferation and reduced the frequency of TNF-α, IL-17 and IFN-γ producing T cells. CONCLUSION: DMF influences the balance between proinflammatory and regulatory T cells, presumably favoring a less proinflammatory environment. DMF also reduces the CNS migratory potential of CD4+ T cells whereas CD8+ T cells are less affected. Altogether, our study suggests an anti-inflammatory effect of DMF mainly on the CD4+ T cell compartment.

Pregnancy-Induced Changes in microRNA Expression in Multiple Sclerosis.

Pregnancy affects the disease course in multiple sclerosis (MS), particularly in the third trimester, where the relapse rate is reduced by as much as two thirds. This study aimed at identifying changes in microRNA (miRNA) and immune cell phenotypes in pregnant MS patients. Discovery and validation studies to detect differentially expressed miRNAs were performed with quantitative real-time PCR on peripheral blood mononuclear cells (PBMC). Flow cytometry analysis was performed on PBMC stained with antibodies directed against surface markers of antigen presenting cells (APCs), NK-cells, NKT cells, CD4+ and CD8+ T cells and subsets of these cell types, including PDL1 and PDL2 expressing subsets. RNA was extracted from whole blood, monocytes, and NK-cells to investigate expression and correlation between regulated miRNAs and mRNAs. In total, 15 miRNAs were validated to be differentially expressed between third trimester pregnant and postpartum MS patients (Benjamini-Hochberg false discovery rate from p = 0.03-0.00004). Of these, 12 miRNAs were downregulated in pregnancy and 6 of the 15 miRNAs were altered by more than ±2-fold (+2.99- to -6.38-fold). Pregnant MS patients had a highly significant increase in the percentage of monocytes and a decrease of NK-cells and myeloid dendritic cells compared to non-pregnant MS patients. We confirm previous reports of a relative increase in CD56-bright NK-cells and a decrease in CD56-dim NK-cells in third trimester of pregnancy and report an increase in non-committed follicular helper cells. PDL1 and PDL2 expression was increased in pregnant patients together with IL10. Also, in monocytes IL10, PDL1, and PDL2 were upregulated whereas miR-1, miR-20a, miR-28, miR-95, miR-146a, miR-335, and miR-625 were downregulated between pregnant and untreated MS patients. IL10, PDL1, and PDL2 were predicted targets of MS pregnancy-changed miRNAs, further supported by their negative correlations. Additionally, previously identified pregnancy-regulated mRNAs were identified as predicted targets of the miRNAs. PDL1 and PDL2 bind PD-1 expressed on T cells with an inhibitory effect on T-cell proliferation and increase in IL10 production. These results indicate that some of the effects behind the disease-ameliorating third trimester of pregnancy might be caused by changed expression of miRNAs and immunoregulatory molecules in monocytes.

Initial high-efficacy disease-modifying therapy in multiple sclerosis: A nationwide cohort study.

OBJECTIVE: To determine the effectiveness of high-efficacy disease-modifying therapies (heDMTs) vs medium-efficacy disease-modifying therapies (meDMT) as the first treatment choice in treatment-naive patients with multiple sclerosis (MS) on disability worsening and relapses. We assessed this using a nationwide population-based MS registry. METHODS: We identified all patients starting a heDMT as first-time treatment from the Danish Multiple Sclerosis Registry and compared treatment outcomes with a propensity score matched sample of patients starting meDMT. RESULTS: We included 388 patients in the study: 194 starting initial therapy with heDMT matched to 194 patients starting meDMT. At 4 years of follow-up, the probabilities of a 6-month confirmed Expanded Disability Status Scale (EDSS) score worsening were 16.7% (95% confidence interval [CI] 10.4%-23.0%) and 30.1% (95% CI 23.1%-37.1%) for heDMT and meDMT initiators, respectively (hazard ratio [HR] 0.53, 95% CI 0.33-0.83, p = 0.006). Patients initiating heDMT also had a lower probability of a first relapse (HR 0.50, 95% CI 0.37-0.67). Results were similar after pairwise censoring and in subgroups with high baseline activity, diagnosis after 2006, or information on baseline T2 lesion load. CONCLUSION: We found a lower probability of 6-month confirmed EDSS score worsening and lower probability of a first relapse in patients starting a heDMT as first therapy, compared to a matched sample starting meDMT. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that for patients with MS, starting heDMT lowers the risk of EDSS worsening and relapses compared to starting meDMT.

Pneumococcal meningitis with normal cerebrospinal biochemistry and no pneumococci at microscopy, mimicking a stroke: a case report.

BACKGROUND: Bacterial meningitis commonly presents with symptoms such as headache, impaired consciousness, neck stiffness, and fever. In most cases, cerebrospinal fluid analysis will yield white cell counts >100/mm3. Atypical presentations occur, especially in the very young or very elderly and the immunocompromised. We report an unusual case of pneumococcal meningitis in a healthy 78-year-old Danish woman who presented with clinical features mimicking a stroke with normal cerebrospinal fluid parameters and without microscopic evidence of bacteria. CASE PRESENTATION: The patient was admitted after being found unconscious on her bed. Upon admittance, she was considered confused, with a temperature of 39.4 °C and slight neutrophilic leukocytosis, but no neck stiffness. A neurological examination revealed bilateral horizontal nystagmus, unstable eye movements, and suspected right-sided gaze paralysis. Cerebrospinal fluid analysis revealed normal parameters, and the microscopy result was negative for bacteria. The most likely diagnosis was considered to be stroke with concomitant infection. However, cerebrospinal fluid and blood cultures subsequently were rapidly positive for pneumococci. Neither immunodeficiency nor blood contamination was considered a likely cause of this discrepancy. CONCLUSIONS: This case emphasizes the need to consider a multidisciplinary approach and empirical meningitis treatment until diagnostic results from microbiological cultures are obtained.

Characterization of naïve, memory and effector T cells in progressive multiple sclerosis.

We characterized naïve, central memory (CM), effector memory (EM) and terminally differentiated effector memory (TEMRA) CD4+ and CD8+ T cells and their expression of CD49d and CD26 in peripheral blood in patients with multiple sclerosis (MS) and healthy controls. CD26+ CD28+ CD4+ TEMRA T cells were increased in all subtypes of MS, and CD26+ CD28+ CD8+ TEMRA T cells were increased in relapsing-remitting and secondary progressive MS. Conversely, in progressive MS, CD49d+ CM T cells were decreased and natalizumab increased the circulating number of all six subsets but reduced the frequency of most subsets expressing CD49d and CD26.

Effect of natalizumab on circulating CD4+ T-cells in multiple sclerosis.

In multiple sclerosis (MS), treatment with the monoclonal antibody natalizumab effectively reduces the formation of acute lesions in the central nervous system (CNS). Natalizumab binds the integrin very late antigen (VLA)-4, expressed on the surface of immune cells, and inhibits VLA-4 dependent transmigration of circulating immune-cells across the vascular endothelium into the CNS. Recent studies suggested that natalizumab treated MS patients have an increased T-cell pool in the blood compartment which may be selectively enriched in activated T-cells. Proposed causes are sequestration of activated T-cells due to reduced extravasation of activated and pro-inflammatory T-cells or due to induction of VLA-4 mediated co-stimulatory signals by natalizumab. In this study we examined how natalizumab treatment altered the distribution of effector and memory T-cell subsets in the blood compartment and if T-cells in general or myelin-reactive T-cells in particular showed signs of increased immune activation. Furthermore we examined the effects of natalizumab on CD4(+) T-cell responses to myelin in vitro. Natalizumab-treated MS patients had significantly increased numbers of effector-memory T-cells in the blood. In T-cells from natalizumab-treated MS patients, the expression of TNF-α mRNA was increased whereas the expression of fourteen other effector cytokines or transcription factors was unchanged. Natalizumab-treated MS patients had significantly decreased expression of the co-stimulatory molecule CD134 on CD4(+)CD26(HIGH) T-cells, in blood, and natalizumab decreased the expression of CD134 on MBP-reactive CD26(HIGH)CD4(+) T-cells in vitro. Otherwise CD4(+) T-cells from natalizumab-treated and untreated MS patients showed similar responses to MBP. In conclusion natalizumab treatment selectively increased the effector memory T-cell pool but not the activation state of T-cells in the blood compartment. Myelin-reactive T-cells were not selectively increased in natalizumab treated MS.

Alterations in KLRB1 gene expression and a Scandinavian multiple sclerosis association study of the KLRB1 SNP rs4763655.

Multiple sclerosis (MS) is a complex autoimmune disease affecting genetically susceptible individuals. A genome-wide association study performed by the International MS Genetics Consortium identified several putative susceptibility genes; among these, the KLRB1 gene is represented by the single-nucleotide polymorphism rs4763655. We could confirm a marginally significant association between rs4763655 and MS (P=0.046, odds ratio=1.06 (1.00-1.13)) in a large Scandinavian case-control study of 5367 MS patients and 4485 controls. The expression of KLRB1 in blood from MS patients was higher compared with healthy controls (P<0.001), and the KLRB1 expression decreased significantly (P<0.001) after interferon (IFN)-ß treatment. KLRB1 was expressed in T and natural killer (NK) cells, and expression mainly decreased in NK cells in patients treated with IFN-ß. Collectively, our results indicate that KLRB1 gene expression is altered in MS and likely to be involved in the pathogenesis of the disease, whereas rs4763655 in KLRB1 seems to have a minimal role in MS susceptibility.

Neocortical and hippocampal neuron and glial cell numbers in the rhesus monkey.

The rhesus monkey is widely used as an experimental animal model in the study of brain function and disease. While previous quantitative studies have provided knowledge of regional numbers, little is known of the total neocortical neuron and glial cell numbers in this species. The aim of this study is to establish quantitative norms. We use the optical fractionator and Cavalieri principle to examine the right hemisphere of eight young rhesus monkeys taken from the control group of an ongoing study. Applying these methods to agar-embedded and vibratome-sectioned tissue, we generate estimates of cell numbers and regional volumes of neocortical and hippocampal regions with coefficients of variance (CV) around 10%. The mean unilateral neocortical neuron number is 1.35 x 10(9) (CV +/- 0.10) and the mean unilateral neocortical glial cell number is 0.78 x 10(9) (CV +/- 0.17). Mean unilateral neocortical volume is found to be 8.5 (CV +/- 0.10) cm(3) after processing, or 19 cm(3) when correcting for shrinkage. The neuron/glia ratio is 1.77. The neurons are distributed with 18% in the frontal cortex, 57% in the temporal and parietal cortices, and 25% in the occipital cortex. In the hippocampal subregions, we found unilateral neuron number of 1.72 x 10(6) (CV +/- 0.13) and glial number of 2.25 x 10(6) (CV +/- 0.17) in CA1, and 0.80 x 10(6) (CV +/- 0.27) neurons and 1.05 x 10(6) (CV +/- 0.26) glial cells in CA2-3. Comparisons with related studies show quantitative variation, but also variations in methods and applications. The results are phylogenetically consistent, apart from the neuron/glia ratio, which is remarkably higher than what is found in other species.